Pyrrole substituted 2-indolinone protein kinase inhibitors

ABSTRACT

The present invention relates to pyrrole substituted 2-indolinone compounds and their pharmaceutically acceptable salts which modulate the activity of protein kinases and therefore are expected to be useful in the prevention and treatment of protein kinase related cellular disorders such as cancer.

CROSS-REFERENCE INFORMATION

This application claims priority under 35 U.S.C. 119(e) to U.S.Provisional Applications Ser. Nos. 60/182,710, filed Feb. 15, 2000,60/216,422 filed on Jul. 6, 2000 and Ser. No. 60/243,532, filed Oct. 27,2000, the disclosures of which are incorporated by reference herein intheir entirety.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to certain 3-pyrrole substituted2-indolinone compounds which modulate the activity of protein kinases(“PKs”). The compounds of this invention are therefore useful intreating disorders related to abnormal PK activity. Pharmaceuticalcompositions comprising these compounds, methods of treating diseasesutilizing pharmaceutical compositions comprising these compounds andmethods of preparing them are also disclosed.

2. State of the Art

The following is offered as background information only and is notadmitted to be prior art to the present invention.

PKs are enzymes that catalyze the phosphorylation of hydroxy groups ontyrosine, serine and threonine residues of proteins. The consequences ofthis seemingly simple activity are staggering; cell growth,differentiation and proliferation, i.e., virtually all aspects of celllife in one way or another depend on PK activity. Furthermore, abnormalPK activity has been related to a host of disorders, ranging fromrelatively non-life threatening diseases such as psoriasis to extremelyvirulent diseases such as glioblastoma (brain cancer).

The PKs can be conveniently broken down into two classes, the proteintyrosine kinases (PTKs) and the serine-threonine kinases (STKs).

One of the prime aspects of PTK activity is their involvement withgrowth factor receptors. Growth factor receptors are cell-surfaceproteins. When bound by a growth factor ligand, growth factor receptorsare converted to an active form which interacts with proteins on theinner surface of a cell membrane. This leads to phosphorylation ontyrosine residues of the receptor and other proteins and to theformation inside the cell of complexes with a variety of cytoplasmicsignaling molecules that, in turn, effect numerous cellular responsessuch as cell division (proliferation), cell differentiation, cellgrowth, expression of metabolic effects to the extracellularmicroenvironment, etc. For a more complete discussion, see Schlessingerand Ullrich, Neuron, 9:303-391 (1992) which is incorporated byreference, including any drawings, as if fully set forth herein.

Growth factor receptors with PTK activity are known as receptor tyrosinekinases (“RTKs”). They comprise a large family of transmembranereceptors with diverse biological activity. At present, at leastnineteen (19) distinct subfamilies of RTKs have been identified. Anexample of these is the subfamily designated the “HER” RTKs, whichinclude EGFR (epithelial growth factor receptor), HER2, HER3 and HER4.These RTKs consist of an extracellular glycosylated ligand bindingdomain, a transmembrane domain and an intracellular cytoplasmiccatalytic domain that can phosphorylate tyrosine residues on proteins.

Another RTK subfamily consists of insulin receptor (IR), insulin-likegrowth factor Ireceptor (IGF-1R) and insulin receptor related receptor(IRR). IR and IGF-1R interact with insulin, IGF-I and IGF-II to form aheteratetramer of two, entirely extracellular glycosylated a subunitsand two β subunits which cross the cell membrane and which contain thetyrosine kinase domain.

A third RTK subfamily is referred to as the platelet derived growthfactor receptor (“PDGFR”) group, which includes PDGFRα, PDGFRβ, CSFIR,c-kit and c-fms. These receptors consist of glycosylated extracellulardomains composed of variable numbers of immunoglobin-like loops and anintracellular domain wherein the tyrosine kinase domain is interruptedby unrelated amino acid sequences.

Another group which, because of its similarity to the PDGFR subfamily,is sometimes subsumed into the later group is the fetus liver kinase(“flk”) receptor subfamily. This group is believed to be made up ofkinase insert domain-receptor fetal liver kinase-1 (KDR/FLK-1, VEGF-R2),flk-1R, flk-4 and fms-like tyrosine kinase 1 (flt-1).

A further member of the tyrosine kinase growth factor receptor family isthe fibroblast growth factor (“FGF”) receptor subgroup. This groupconsists of four receptors, FGFR1-4, and seven ligands, FGF1-7. Whilenot yet well defined, it appears that the receptors consist of aglycosylated extracellular domain containing a variable number ofimmunoglobin-like loops and an intracellular domain in which thetyrosine kinase sequence is interrupted by regions of unrelated aminoacid sequences.

Still another member of the tyrosine kinase growth factor receptorfamily is the vascular endothelial growth factor (VEGF”) receptorsubgroup. VEGF is a dimeric glycoprotein similar to PDGF but hasdifferent biological functions and target cell specificity in vivo. Inparticular, VEGF is presently thought to play an essential role isvasculogenesis and angiogenesis.

A more complete listing of the known RTK subfamilies is described inPlowman et al., DN&P, 7(6):334-339 (1994) which is incorporated byreference, including any drawings, as if fully set forth herein.

In addition to the RTKs, there also exists a family of entirelyintracellular PTKs called “non-receptor tyrosine kinases” or “cellulartyrosine kinases.” This latter designation, abbreviated “CTK,” will beused herein. CTKs do not contain extracellular and transmembranedomains. At present, over 24 CTKs in 11 subfamilies (Src, Frk, Btk, Csk,Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have been identified. The Srcsubfamily appear so far to be the largest group of CTKs and includesSrc, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. For a more detaileddiscussion of CTKs, see Bolen, Oncogene, 8:2025-2031 (1993), which isincorporated by reference, including any drawings, as if fully set forthherein.

The serine/threonine kinases, STKs, like the CTKs, are predominantlyintracellular although there are a few receptor kinases of the STK type.STKs are the most common of the cytosolic kinases; i.e., kinases thatperform their function in that part of the cytoplasm other than thecytoplasmic organelles and cytoskelton. The cytosol is the region withinthe cell where much of the cell's intermediary metabolic andbiosynthetic activity occurs; e.g., it is in the cytosol that proteinsare synthesized on ribosomes.

RTKs, CTKs and STKs have all been implicated in a host of pathogenicconditions including, significantly, cancer. Other pathogenic conditionswhich have been associated with PTKs include, without limitation,psoriasis, hepaticcirrhosis, diabetes, angiogenesis, restenosis, oculardiseases, rheumatoid arthritis and other inflammatory disorders,immunological disorders such as autoimmune disease, cardiovasculardisease such as atherosclerosis and a variety of renal disorders.

With regard to cancer, two of the major hypotheses advanced to explainthe excessive cellular proliferation that drives tumor developmentrelate to functions known to be PK regulated. That is, it has beensuggested that malignant cell growth results from a breakdown in themechanisms that control cell division and/or differentiation. It hasbeen shown that the protein products of a number of proto-oncogenes areinvolved in the signal transduction pathways that regulate cell growthand differentiation. These protein products of proto-oncogenes includethe extracellular growth factors, transmembrane growth factor PTKreceptors (RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs, discussedabove.

In view of the apparent link between PK-related cellular activities andwide variety of human disorders, it is no surprise that a great deal ofeffort is being expended in an attempt to identify ways to modulate PKactivity. Some of this effort has involved biomimetic approaches usinglarge molecules patterned on those involved in the actual cellularprocesses. (e.g., mutant ligands (U.S. Pat. No. 4,966,849); solublereceptors and antibodies (App. No. WO 94/10202, Kendall and Thomas,Proc. Nat'l Acad. Sci., 90:10705-09 (1994), Kim, et al., Nature,362:841-844 (1993)); RNA ligands (Jelinek, et al., Biochemistry,33:10450-56); Takano, et al., Mol. Bio. Cell 4:358A (1993); Kinsella, etal., Exp. Cell Res. 199:56-62 (1992); Wright, et al., J. Cellular Phys.,152:448-57) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al., Proc.Am. Assoc. Cancer Res., 35:2268 (1994)).

In addition to the above, attempts have been made to identify smallmolecules which act as PK inhibitors. For example, bis-monocylic,bicyclic and heterocyclic aryl compounds (PCT WO 92/20642),vinyleneazaindole derivatives (PCT WO 94/14808) and1-cyclopropyl-4-pyridylquinolones (U.S. Pat. No. 5,330,992) have beendescribed as tyrosine kinase inhibitors. Styryl compounds (U.S. Pat. No.5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No.5,302,606), quinazoline derivatives (EP App. No.0 566 266 A1),selenaindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxyliccompounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO91/15495) have all been described as PTK inhibitors useful in thetreatment of cancer.

SUMMARY OF THE INVENTION

The present invention is directed to certain 3-pyrrole substituted2-indolinone compounds which exhibit PK modulating ability and aretherefore useful in treating disorders related to abnormal PK activity.

Accordingly, in one aspect, the present invention relates to 3-pyrrolesubstituted 2-indolinones of Formula (I):

wherein:

-   -   R¹ is selected from the group consisting of hydrogen, halo,        alkyl, cyclkoalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy,        alkoxy, —(CO)R¹⁵, —NR¹³R¹⁴, —(CH₂)_(r)R¹⁶ and —C(O)NR⁸R⁹;    -   R² is selected from the group consisting of hydrogen, halo,        alkyl, trihalomethyl, hydroxy, alkoxy, cyano, —NR¹³R¹⁴,        —NR¹³C(O)R¹⁴, —C(O) R¹⁵, aryl, heteroaryl, —S(O)₂NR¹³R¹⁴ and        —SO₂R²⁰ (wherein R²⁰ is alkyl, aryl, aralkyl, heteroaryl and        heteroaralkyl);    -   R³ is selected from the group consisting of hydrogen, halogen,        alkyl, trihalomethyl, hydroxy, alkoxy, —(CO)R¹⁵, —NR¹³R¹⁴, aryl,        heteroaryl, —NR¹³S(O)₂R^(14,) —S(O)₂NR¹³R¹⁴, —NR¹³C(O)R¹⁴,    -   —NR¹³C(O)OR¹⁴ and —SO₂R²⁰ (wherein R²⁰ is alkyl, aryl, aralkyl,        heteroaryl and heteroaralkyl);    -   R⁴ is selected from the group consisting of hydrogen, halogen,        alkyl, hydroxy, alkoxy and —NR¹³R¹⁴;    -   R⁵ is selected from the group consisting of hydrogen, alkyl and        —C(O)R¹⁰;    -   R⁶ is selected from the group consisting of hydrogen, alkyl and        —C(O)R¹⁰;    -   R⁷ is selected from the group consisting of hydrogen, alkyl,        aryl, heteroaryl, —C(O)R¹⁷ and —C(O)R¹⁰; or    -   R⁶ and R⁷ may combine to form a group selected from the group        consisting of —(CH₂)₄—, —(CH₂)₅— and —(CH₂)₆—; with the proviso        that at least one of R⁵, R⁶ or R⁷ must be —C(O)R¹⁰;    -   R⁸ and R⁹ are independently selected from the group consisting        of hydrogen, alkyl and aryl;    -   R¹⁰ is selected from the group consisting of hydroxy, alkoxy,        aryloxy, —N(R¹¹)(CH₂)_(n)R¹², and —NR¹³R¹⁴;    -   R¹¹ is selected from the group consisting of hydrogen and alkyl;    -   R¹² is selected from the group consisting of —NR¹³R¹⁴, hydroxy,        —C(O)R¹⁵, aryl, heteroaryl, —N⁺(O⁻)R¹³R¹⁴, —N(OH)R¹³, and        —NHC(O)R^(a) (wherein R^(a) is unsubstituted alkyl, haloalkyl,        or aralkyl);    -   R¹³ and R¹⁴ are independently selected from the group consisting        of hydrogen, alkyl, cyanoalkyl, cycloalkyl, aryl and heteroaryl;        or    -   R¹³ and R¹⁴ may combine to form a heterocyclo group;    -   R¹⁵ is selected from the group consisting of hydrogen, hydroxy,        alkoxy and aryloxy;    -   R¹⁶ is selected from the group consisting of hydroxy, —C(O)R¹⁵,        —NR¹³R¹⁴ and —C(O)NR¹³R¹⁴;    -   R¹⁷ is selected from the group consisting of alkyl, cycloalkyl,        aryl and heteroaryl;    -   R²⁰ is alkyl, aryl, aralkyl or heteroaryl; and    -   n and r are independently 1, 2, 3, or 4;        or a pharmaceutically acceptable salt thereof.

Preferably, R¹ is selected from the group consisting of hydrogen, halo,alkyl, cyclkoalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,—C(O)R¹⁵, —NR¹³R¹⁴, —(CH₂)_(r)R¹⁶ and —C(O)NR⁸R⁹;

-   -   R² is selected from the group consisting of hydrogen, halo,        alkyl, trihalomethyl, hydroxy, alkoxy, —NR¹³R¹⁴, —NR¹³C(O)R¹⁴,        —C(O)R¹⁵, aryl, heteroaryl, and —S(O)₂NR¹³R¹⁴;    -   R³ is selected from the group consisting of hydrogen, halogen,        alkyl, trihalomethyl, hydroxy, alkoxy, —(CO)R¹⁵, —NR¹³R¹⁴, aryl,        heteroaryl, —NR¹³S(O)₂R^(14,) —S(O)₂NR¹³R¹⁴, —NR¹³C(O)R¹⁴and        —NR¹³C(O)OR¹⁴;    -   R⁴ is selected from the group consisting of hydrogen, halogen,        alkyl, hydroxy, alkoxy and —NR¹³R¹⁴;    -   R⁵ is selected from the group consisting of hydrogen, alkyl and        —C(O)R¹⁰;

R⁶ selected from the group consisting of hydrogen, alkyl and —C(O)R¹⁰;

R⁷ is selected from the group consisting of hydrogen, alkyl, aryl,heteroaryl, —C(O)R¹⁷ and —C(O)R¹⁰;

R⁶ and R⁷ may combine to form a group selected from the group consistingof —(CH₂)₄—, —(CH₂)₅— and —(CH₂)₆—;

-   -   with the proviso that at least one of R⁵, R⁶ or R⁷ must be        —C(O)R¹⁰;    -   R⁸ and R⁹ are independently selected from the group consisting        of hydrogen, alkyl and aryl;    -   R¹⁰ is selected from the group consisting of hydroxy, alkoxy,        aryloxy, —N(R¹¹)(CH₂)_(n)R¹² and —NR¹³R¹⁴;    -   R¹¹ is selected from the group consisting of hydrogen and alkyl;    -   R¹² is selected from the group consisting of —NR¹³R¹⁴, hydroxy,        —C(O)R¹⁵, aryl and heteroaryl;    -   R¹³ and R¹⁴ are independently selected from the group consisting        of hydrogen, alkyl, cycloalkyl, aryl and heteroaryl;    -   R¹³ and R¹⁴ may combine to form a group selected from the group        consisting of —(CH₂)₄—, —(CH₂)₅—, —(CH₂)₂O(CH₂)₂—, and        —(CH₂)₂N(CH₃)(CH₂)₂—;    -   R¹⁵ is selected from the group consisting of hydrogen, hydroxy,        alkoxy and aryloxy;    -   R¹⁶ is selected from the group consisting of hydroxy, —C(O)R¹⁵,        —NR¹³R¹⁴ and —C(O)NR¹³R¹⁴;    -   R¹⁷ is selected from the group consisting of alkyl, cycloalkyl,        aryl and heteroaryl; and    -   n and r are independently 1, 2, 3, or 4;        or a pharmaceutically acceptable salt thereof.

In a second aspect this invention is directed to a pharmaceuticalcomposition comprising one or more compound(s) of Formula (I) or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable excipient.

In a third aspect, this invention is directed to a method of treatingdiseases mediated by abnormal protein kinase activity, in particular,receptor tyrosine kinases (RTKs), non-receptor protein tyrosine kinases(CTKs) and serine/threonine protein kinases (STKs), in an organism, inparticular humans, which method comprises administering to said organisma pharmaceutical composition comprising a compound of Formula (I). Suchdiseases include by way of example and not limitation, cancer, diabetes,hepatic cirrhosis, cardiovasacular disease such as atherosclerosis,angiogenesis, immunological disease such as autoimmune disease and renaldisease.

In a fourth aspect, this invention is directed to a method of modulatingof the catalytic activity of PKs, in particular, receptor tyrosinekinases (RTKs), non-receptor protein tyrosine kinases (CTKs) andserine/threonine protein kinases (STKs), using a compound of thisinvention which may be carried out in vitro or in vivo. In particular,the receptor protein kinase whose catalytic activity is modulated by acompound of this invention is selected from the group consisting of EGF,HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRα, PDGFRβ, CSFIR, C-Kit, C-fms,Flk-1R, Flk4, KDR/Flk-1, Flt-1 , FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R.The cellular tyrosine kinase whose catalytic activity is modulated by acompound of this invention is selected from the group consisting of Src,Frk, Btk, Csk, Abl, ZAP70, Fes/Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck,Blk, Hck, Fgr and Yrk. The serine-threonine protein kinase whosecatalytic activity is modulated by a compound of this invention isselected from the group consisting of CDK2 and Raf.

In a fifth aspect, this invention is directed to the use of a compoundof Formula (I) in the preparation of a medicament which is useful in thetreatment of a disease mediated by abnormal PK activity.

In a sixth aspect, this invention is directed to an intermediate ofFormula (II):

wherein:

-   -   R⁵ is selected from the group consisting of hydrogen, alkyl and        —C(O)R¹⁰;    -   R⁶ is selected from the group consisting of hydrogen, alkyl and        —C(O)R¹⁰;    -   R⁷ is selected from the group consisting of hydrogen, alkyl,        aryl, heteroaryl, —C(O)R¹⁷ and —C(O)R¹⁰;    -   R⁶ and R⁷ may combine to form a group selected from the group        consisting of —(CH₂)₄—, —(CH₂)₅— and —(CH₂)₆—;    -   with the proviso that at least one of R⁵, R⁶ or R⁷ must be        —C(O)R¹⁰;    -   R¹⁰ is selected from the group consisting of hydroxy, alkoxy,        aryloxy, —N (R¹¹)(CH₂)_(n)R¹² and —NR¹³R¹⁴;    -   R¹¹ is selected from the group consisting of hydrogen and        alkyl;,    -   R¹² is selected from the group consisting of —NR¹³R¹⁴, hydroxy,        —C(O)R¹⁵, aryl and heteroaryl;    -   R¹³ and R¹⁴ are independently selected from the group consisting        of hydrogen, alkyl, cyanoalkyl, cycloalkyl, aryl and heteroaryl;        or    -   R¹³ and R¹⁴ may combine to form a heterocyclo group;    -   R¹⁵ is selected from the group consisting of hydrogen, hydroxy,        alkoxy and aryloxy;    -   R¹⁷ is selected from the group consisting of alkyl, cycloalkyl,        aryl and heteroaryl; and    -   n is 1, 2, 3, or 4.

Preferaby, R⁵ or R⁶, in the compound of formula II, is —C(O)R¹⁰;

-   -   R⁶ is selected from the group consisting of hydrogen, and alkyl,        more preferably hydrogen or methyl;    -   R⁵ is selected from the group consisting of hydrogen, and alkyl,        more preferably hydrogen or methyl when R⁶ is —COR¹⁰;    -   R⁶ is selected from the group consisting of hydrogen, and alkyl,        more preferably hydrogen or methyl when R⁵ is —COR¹⁰;;    -   R⁷ is selected from the group consisting of hydrogen, alkyl, and        aryl, more preferably hydrogen, methyl or phenyl;    -   R¹⁰ is selected from the group consisting of hydroxy, alkoxy,        —N(R¹¹)(CH₂)_(n)R¹² and —NR¹³R¹⁴;    -   R¹¹ is selected from the group consisting of hydrogen and alkyl,        more preferably hydrogen or methyl;    -   R¹² is selected from the group consisting of —NR¹³R¹⁴;    -   R¹³ and R¹⁴ are independently selected from the group consisting        of hydrogen, or alkyl; or    -   R¹³ and R¹⁴ may combine to form a heterocyclo group; and    -   n is 1, 2 or 3.

Within the above preferred groups, more preferred groups of intermediatecompounds are those wherein R⁵, R⁶, R¹¹, R¹², R¹³ or R¹⁴ areindependently groups described in the section titled “preferredembodiments” herein below.

In a seventh aspect, this invention is directed to methods of preparingcompounds,of Formula (I).

Lastly, this invention is also directed to identifying a chemicalcompound that modulates the catalytic activity of a protein kinase bycontacting cells expressing said protein kinase with a compound or asalt of the present invention and then monitoring said cells for aneffect.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

Unless otherwise stated the following terms used in the specificationand claims have the meanings discussed below:

“Alkyl” refers to a saturated aliphatic hydrocarbon radical includingstraight chain and branched chain groups of 1 to 20 carbon atoms(whenever a numerical range; e.g. “1-20”, is stated herein, it meansthat the group, in this case the alkyl group, may contain 1 carbon atom,2 carbon atoms, 3 carbon atoms, etc. up to and including 20 carbonatoms). Alkyl groups containing from 1 to 4 carbon atoms are refered toas lower alkyl groups. When said lower alkyl groups lack substituents,they are referred to as unsubstituted lower alkyl groups. Morepreferably, an alkyl group is a medium size alkyl having 1 to 10 carbonatoms e.g., methyl, ethyl, propyl, 2-propyl, n-butyl, iso-butyl,tert-butyl, pentyl, and the like. Most preferably, it is a lower alkylhaving 1 to 4 carbon atoms e.g., methyl, ethyl, propyl, 2-propyl,n-butyl, iso-butyl, or tert-butyl, and the like. The alkyl group may besubstituted or unsubstituted. When substituted, the substituent group(s)is preferably one or more, more preferably one to three, even morepreferably one or two substituent(s) independently selected from thegroup consisting of halo, hydroxy, unsubstituted lower alkoxy, aryloptionally substituted with one or more groups, preferably one, two orthree groups which are independently of each other halo, hydroxy,unsubstituted lower alkyl or unsubstituted lower alkoxy groups, aryloxyoptionally substituted with one or more groups, preferably one, two orthree groups which are independently of each other halo, hydroxy,unsubstituted lower alkyl or unsubstituted lower alkoxy groups, 6-memberheteroaryl having from 1 to 3 nitrogen atoms in the ring, the carbons inthe ring being optionally substituted with one or more groups,preferably one, two or three groups which are independently of eachother halo, hydroxy, unsubstituted lower alkyl or unsubstituted loweralkoxy groups, 5-member heteroaryl having from 1 to 3 heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur, thecarbon and the nitrogen atoms in the group being optionally substitutedwith one or more groups, preferably one, two or three groups which areindependently of each other halo, hydroxy, unsubstituted lower alkyl orunsubstituted lower alkoxy groups, 5- or 6-member heteroalicyclic grouphaving from 1 to 3 heteroatoms selected from the group consisting ofnitrogen, oxygen and sulfur, the carbon and nitrogen (if present) atomsin the group being optionally substituted with one or more groups,preferably one, two or three groups which are independently of eachother halo, hydroxy, unsubstituted lower alkyl or unsubstituted loweralkoxy groups, mercapto, (unsubstituted lower alkyl)thio, arylthiooptionally substituted with one or more groups, preferably one, two orthree groups which are independently of each other halo, hydroxy,unsubstituted lower alkyl or unsubstituted lower alkoxy groups, cyano,acyl, thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido, R¹⁸S(O)—,R¹⁸S(O)₂—, —C(O)OR¹⁸, R¹⁸C(O)O—, and —NR¹⁸R¹⁹, wherein R¹⁸ and R¹⁹ areindependently selected from the group consisting of hydrogen,unsubstituted lower alkyl, trihalomethyl, unsubstituted(C₃-C₆)cycloalkyl, unsubstituted lower alkenyl, unsubstituted loweralkynyl and aryl optionally substituted with one or more, groups,preferably one, two or three groups which are independently of eachother halo, hydroxy, unsubstituted lower alkyl or unsubstituted loweralkoxy groups.

Preferably, the alkyl group is substituted with one or two substituentsindependently selected from the group consisting of hydroxy, 5- or6-member heteroalicyclic group having from 1 to 3 heteroatoms selectedfrom the group consisting of nitrogen, oxygen and sulfur, the carbon andnitrogen (if present) atoms in the group being optionally substitutedwith one or more groups, preferably one, two or three groups which areindependently of each other halo, hydroxy, unsubstituted lower alkyl orunsubstituted lower alkoxy groups, 5-member heteroaryl having from 1 to3 heteroatoms selected from the group consisting of nitrogen, oxygen andsulfur, the carbon and the nitrogen atoms in the group being optionallysubstituted with one or more groups, preferably one, two or three groupswhich are independently of each other halo, hydroxy, unsubstituted loweralkyl or unsubstituted lower alkoxy groups, 6-member heteroaryl havingfrom 1 to 3 nitrogen atoms in the ring, the carbons in the ring beingoptionally substituted with one or more groups, preferably one, two orthree groups which are independently of each other halo, hydroxy,unsubstituted lower alkyl or unsubstituted lower alkoxy groups, or—NR¹⁸R¹⁹, wherein R¹⁸ and R¹⁹ are independently selected from the groupconsisting of hydrogen, unsubstituted lower alkyl. Even more preferablythe alkyl group is substituted with one or two substituents which areindependently of each other hydroxy, dimethylamino, ethylamino,diethylamino, dipropylamino, pyrrolidino, piperidino, morpholino,piperazino, 4-lower alkylpiperazino, phenyl, imidazolyl, pyridinyl,pyridazinyl, pyrimidinyl, oxazolyl, triazinyl, and the like.

“Cycloalkyl” refers to a 3 to 8 member all-carbon monocyclic ring, anall-carbon 5-member/6-member or 6-member/6-member fused bicyclic ring ora multicyclic fused ring (a “fused” ring system means that each ring inthe system shares an adjacent pair of carbon atoms with each other ringin the system) group wherein one or more of the rings may contain one ormore double bonds but none of the rings has a completely conjugatedpi-electron system.

Examples, without limitation, of cycloalkyl groups are cyclopropane,cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene,adamantane, cycloheptane, cycloheptatriene, and the like. A cycloalkylgroup may be substituted or unsubstituted. When substituted, thesubstituent group(s) is preferably one or more, more preferably one ortwo substituents, independently selected from the group consisting ofunsubstituted lower alkyl, trihaloalkyl, halo, hydroxy, unsubstitutedlower alkoxy, aryl optionally substituted with one or more, preferablyone or two groups independently of each other halo, hydroxy,unsubstituted lower alkyl or unsubstituted lower alkoxy groups, aryloxyoptionally substituted with one or more, preferably one or two groupsindependently of each other halo, hydroxy, unsubstituted lower alkyl orunsubstituted lower alkoxy groups, 6-member heteroaryl having from 1 to3 nitrogen atoms in the ring, the carbons in the ring being optionallysubstituted with one or more, preferably one or two groups independentlyof each other halo, hydroxy, unsubstituted lower alkyl or unsubstitutedlower alkoxy groups, 5-member heteroaryl having from 1 to 3 heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur, thecarbon and nitrogen atoms of the group being optionally substituted withone or more, preferably one or two groups independently of each otherhalo, hydroxy, unsubstituted lower alkyl or unsubstituted lower alkoxygroups, 5- or 6-member heteroalicyclic group having from 1 to 3heteroatoms selected from the group consisting of nitrogen, oxygen andsulfur, the carbon and nitogen (if present)atoms in the group beingoptionally substituted with one or more, preferably one or two groupsindependently of each other halo, hydroxy, unsubstituted lower alkyl orunsubstituted lower alkoxy groups, mercapto,(unsubstituted loweralkyl)thio, arylthio optionally substituted with one or more, preferablyone or two groups independently of each other halo, hydroxy,unsubstituted lower alkyl or unsubstituted lower alkoxy groups, cyano,acyl, thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido, R¹⁸S(O)—,R¹⁸S(O)₂—, —C(O)OR¹⁸, R¹⁸C(O)O—, and —NR¹⁸R¹⁹ are as defined above.

“Alkenyl” refers to a lower alkyl group, as defined herein, consistingof at least two carbon atoms and at least one carbon-carbon double bond.Representative examples include, but are not limited to, ethenyl,1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, and the like.

“Alkynyl” refers to a lower alkyl group, as defined herein, consistingof at least two carbon atoms and at least one carbon-carbon triple bond.Representative examples include, but are not limited to, ethynyl,1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl, and the like.

“Aryl” refers to an all-carbon monocyclic or fused-ring polycyclic(i.e., rings which share adjacent pairs of carbon atoms) groups of 1 to12 carbon atoms having a completely conjugated pi-electron system.Examples, without limitation, of aryl groups are phenyl, naphthalenyland anthracenyl. The aryl group may be substituted or unsubstituted.When substituted, the substituted group(s) is preferably one or more,more preferably one, two or three, even more preferably one or two,independently selected from the group consisting of unsubstituted loweralkyl, trihaloalkyl, halo, hydroxy, unsubstituted lower alkoxy,mercapto,(unsubstituted lower alkyl)thio, cyano, acyl, thioacyl,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, nitro, N-sulfonamido, S-sulfonamido, R¹⁸S(O)—, R¹⁸S(O)₂—,—C(O)OR¹⁸, R¹⁸C(O)O—, and —NR¹⁸R¹⁹, with R¹⁸ and R¹⁹ as defined above.Preferably, the aryl group is optionally substituted with one or twosubstituents independently selected from halo, unsubstituted loweralkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono ordialkylamino, carboxy, or N-sulfonamido.

“Heteroaryl” refers to a monocyclic or fused ring (i.e., rings whichshare an adjacent pair of atoms) group of 5 to 12 ring atoms containingone, two, or three ring heteroatoms selected from N, O, or S, theremaining ring atoms being C, and, in addition, having a completelyconjugated pi-electron system. Examples, without limitation, ofunsubstituted heteroaryl groups are pyrrole, furan, thiophene,imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline,isoquinoline, purine and carbazole. The heteroaryl group may besubstituted or unsubstituted. When substituted, the substituted group(s)is preferably-one or more, more preferably one, two, or three, even morepreferably one or two, independently selected from the group consistingof unsubstituted lower alkyl, trihaloalkyl, halo, hydroxy, unsubstitutedlower alkoxy, mercapto, (unsubstituted lower alkyl)thio, cyano, acyl,thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido, R¹⁸S(O)—,R¹⁸O)₂—, —C(O)OR¹⁸, R¹⁸C(O)O—, and —NR¹⁸R¹⁹, with R¹⁸ and R¹⁹ as definedabove. Preferably, the heteroaryl group is optionally substituted withone or two substituents independently selected from halo, unsubstitutedlower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono ordialkylamino, carboxy, or N-sulfonamido.

“Heteroalicyclic” refers to a monocyclic or fused ring group having inthe ring(s) of 5 to 9 ring atoms in which one or two ring atoms areheteroatoms selected from N, O, or S(O)_(n) (where n is an integer from0 to 2), the remaining ring atoms being C. The rings may also have oneor more double bonds. However, the rings do not have a completelyconjugated pi-electron system. Examples, without limitation, ofunsubstituted heteroalicyclic groups are pyrrolidino, piperidino,piperazino, morpholino, thiomorpholino, homopiperazino, and the like.The heteroalicyclic ring may be substituted or unsubstituted. Whensubstituted, the substituted group(s) is preferably one or more, morepreferably one, two or three, even more preferably one or two,independently selected from the group consisting of unsubstituted loweralkyl, trihaloalkyl, halo, hydroxy, unsubstituted lower alkoxy,mercapto,(unsubstituted lower alkyl)thio, cyano, acyl, thioacyl,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, nitro, N-sulfonamido, S-sulfonamido, R¹⁸S(O)—, R¹⁸S(O)₂—,—C(O)OR¹⁸, R¹⁸C(O)O—, and —NR¹⁸R¹⁹, with R¹⁸ and R¹⁹ as defined above.Preferably, the heteroalicyclic group is optionally substituted with oneor two substituents independently selected from halo, unsubstitutedlower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono ordialkylamino, carboxy, or N-sulfonamido.

Preferably, the heteroalicyclic group is optionally substituted with oneor two substituents independently selected from halo, unsubstitutedlower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono ordialkylamino, carboxy, or N-sulfonamido.

“Heterocycle” means a saturated cyclic radical of 3 to 8 ring atoms inwhich one or two ring atoms are heteroatoms selected from N, O, orS(O)_(n) (where n is an integer from 0 to 2), the remaining ring atomsbeing C, where one or two C atoms may optionally be replaced by acarbonyl group. The heterocyclyl ring may be optionally substitutedindependently with one, two, or three substituents selected fromoptionally substituted lower alkyl (substituted with 1 or 2 substituentsindependently selected from carboxy or ester), haloalkyl, cyanoalkyl,halo, nitro, cyano, hydroxy, alkoxy, amino, monoalkylamino,dialkylamino, aralkyl, heteroaralkyl, —COR (where R is alkyl) or

COOR where R is (hydrogen or alkyl). More specifically the termheterocyclyl includes, but is not limited to, tetrahydropyranyl,2,2-dimethyl-1,3-dioxolane, piperidino, N-methylpiperidin-3-yl,piperazino, N-methylpyrrolidin-3-yl, 3-pyrrolidino, morpholino,thiomorpholino, thiomorpholino-1-oxide, thiomorpholino-1,1-dioxide,4-ethyloxycarbonylpiperazino, 3-oxopiperazino, 2-imidazolidone,2-pyrrolidinone, 2-oxohomopiperazino, tetrahydropyrimidin-2-one, and thederivatives thereof. Preferably, the heterocycle group is optionallysubstituted with one or two substituents independently selected fromhalo, unsubstituted lower alkyl, lower alkyl substituted with carboxy,ester hydroxy, mono or dialkylamino.

“Hydroxy” refers to an —OH group.

“Alkoxy” refers to both an —O-(unsubstituted alkyl) and an—O-(unsubstituted cycloalkyl) group. Representative examples include,but are not limited to, e.g., methoxy, ethoxy, propoxy, butoxy,cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and thelike.

“Aryloxy” refers to both an —O-aryl .and an —O-heteroaryl group, asdefined herein. Representative examples include, but are not limited to,phenoxy, pyridinyloxy, furanyloxy, thienyloxy, pyrimidinyloxy,pyrazinyloxy, and the like, and derivatives thereof.

“Mercapto” refers to an —SH group.

“Alkylthio” refers to both an —S-(unsubstituted alkyl) and an—S-(unsubstituted cycloalkyl) group. Representative examples include,but are not limited to, e.g., methylthio, ethylthio, propylthio,butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio,cyclohexylthio, and the like.

“Arylthio” refers to both an —S-aryl and an —S-heteroaryl group, asdefined herein. Representative examples include, but are not limited to,phenylthio, pyridinylthio, furanylthio, thientylthio, pyrimidinylthio,and the like and derivatives thereof.

“Acyl” refers to a —C(O)—R″ group, where R″ is selected from the groupconsisting of hydrogen, unsubstituted lower alkyl, trihalomethyl,unsubstituted cycloalkyl, aryl optionally substituted with one or more,preferably one, two, or three substituents selected from the groupconsisting of unsubstituted lower alkyl, trihalomethyl, unsubstitutedlower alkoxy, halo and —NR¹⁸R¹⁹ groups; heteroaryl (bonded through aring carbon) optionally substituted with one or more, preferably one,two, or three substitutents selected from the group consisting ofunsubstituted lower alkyl, trihaloalkyl, unsubstituted lower alkoxy,halo and —NR¹⁸R¹⁹ groups and heteroalicyclic (bonded through a ringcarbon) optionally substituted with one or more, preferably one, two, orthree substituents selected from the group consisting of unsubstitutedlower alkyl, trihaloalkyl, unsubstituted lower alkoxy, halo and —NR¹⁸R¹⁹groups. Representative acy groups include, but are not limited to,acetyl, trifluoroacetyl, benzoyl, and the like

“Aldehyde” refers to an acyl group in which R″ is hydrogen.

“Thioacyl” refers to a —C(S)—R″ group, with R″ as defined herein.

“Ester” refers to a —C(O)O—R″ group with R″ as defined herein exceptthat R″ cannot be hydrogen.

“Acetyl” group refers to a —C(O)CH₃ group.

“Halo” group refers to fluorine, chlorine, bromineor iodine, preferablyfluorine or chlorine.

“Trihalomethyl” group refers to a —CX₃ group wherein X is a halo groupas defined herein.

“Trihalomethanesulfonyl” group refers to a X₃CS(═O)₂— groups with X asdefined above.

“Cyano” refers to a —C≡N group.

“Methylenedioxy” refers to a —OCH₂O— group where the two oxygen atomsare bonded to adjacent carbon atoms.

“Ethylenedioxy” group refers to a —OCH₂CH₂O— where the two oxygen atomsare bonded to adjacent carbon atoms.

“S-sulfonamido” refers to a —S(O)₂NR¹⁸R¹⁹ group, with R¹⁸ and R¹⁹ asdefined herein.

“N-sulfonamido” refers to a —NR¹⁸S(O)₂R¹⁹ group, with R¹⁸ and R¹⁹ asdefined herein.

“O-carbamyl” group refers to a —OC(O)NR¹⁸R¹⁹ group with R¹⁸ and R¹⁹ asdefined herein.

“N-carbamyl” refers to an R¹⁸OC(O)NR¹⁹— group, with R¹⁸ and R¹⁹ asdefined herein.

“O-thiocarbamyl” refers to a —OC(S)NR¹⁸R¹⁹ group with R¹⁸ and R¹⁹ asdefined herein.

“N-thiocarbamyl” refers to a R¹⁸OC(S)NR¹⁹— group, with R¹⁸ and R¹⁹ asdefined herein.

“Amino” refers to an —NR¹⁸R¹⁹ group, wherein R¹⁸ and R¹⁹ are bothhydrogen.

“C-amido” refers to a —C(O)NR¹⁸R¹⁹ group with R¹⁸ and R¹⁹ as definedherein.

“N-amido” refers to a R¹⁸C(O)NR¹⁹— group, with R¹⁸ and R¹⁹ as definedherein.

“Nitro” refers to a —NO₂ group.

“Haloalkyl” means an unsubstituted alkyl, preferably unsubstituted loweralkyl as defined above that is substituted with one or more same ordifferent halo atoms, e.g., —CH₂Cl, —CF₃, —CH₂CF₃, —CH₂CCl₃, and thelike.

“Aralkyl” means unsubstituted alkyl, preferably unsubstituted loweralkyl as defined above which is substituted with an aryl group asdefined above, e.g., —CH₂phenyl, —(CH₂)₂phenyl, —(CH₂)₃phenyl,CH₃CH(CH₃)CH₂phenyl, and the like and derivatives thereof.

“Heteroaralkyl” group means unsubstituted alkyl, preferablyunsubstituted lower alkyl as defined above which is substituted with aheteroaryl group, e.g., —CH₂pyridinyl, —(CH₂)₂pyrimidinyl,—(CH₂)₃imidazolyl, and the like, and derivatives thereof.

“Monoalkylamino” means a radical —NHR where R is an unsubstitued alkylor unsubstituted cycloalkyl group as defined above, e.g., methylamino,(1-methylethyl)amino, cyclohexylamino, and the like.

“Dialkylamino” means a radical —NRR where each R is independently anunsubstitued alkyl or unsubstituted cycloalkyl group as defined above,e.g., dimethylamino, diethylamino, (1-methylethyl)-ethylamino,cyclohexylmethylamino, cyclopentylmethylamino, and the like.

“Cyanoalkyl” means unsubstituted alkyl, preferably unsubstituted loweralkyl as defined above, which is substituted with 1 or 2 cyano groups.

“Optional” or “optionally” means that the subsequently described eventor circumstance may but need not occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not. For example, “heterocycle group optionallysubstituted with an alkyl group” means that the alkyl may but need notbe present, and the description includes situations where theheterocycle group is substituted with an alkyl group and situationswhere the heterocyclo group is not substituted with the alkyl group.

The terms “2-indolinone”, “indolin-2-one” and “2-oxindole” are usedinterchangeably herein to refer to a molecule having the chemicalstructure:

The term “pyrrole” refers to a molecule having the chemical structure:

The term “pyrrole substituted 2-indolinone” and“3-pyrrolidenyl-2-indolinone” are used interchangeably herein to referto a chemical compound having the general structure shown in Formula(I).

Compounds that have the same molecular formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers”. Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers”. Stereoisomers that arenot mirror images of one another are termed “diastereomers” and thosethat are non-superimposable mirror images of each other are termed“enantiomers”. When a compound has an asymmetric center, for example, itis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCahn and Prelog, or by the manner in which the molecule rotates theplane of polarized light and designated as dextrorotatory orlevorotatory (i.e., as (+) or (−)-isomers respectively). A chiralcompound can exist as either individual enantiomer or as a mixturethereof. A mixture containing equal proportions of the enantiomers iscalled a “racemic mixture”.

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof. For example, if the R⁶substituent in a compound of formula (I) is 2-hydroxyethyl, then thecarbon to which the hydroxy group is attached is an asymmetric centerand therefore the compound of Formula (I) can exist as an (R)- or(S)-stereoisomer. Unless indicated otherwise, the description or namingof a particular compound in the specification and claims is intended toinclude both individual enantiomers and mixtures, racemic or otherwise,thereof. The methods for the determination of stereochemistry and theseparation of stereoisomers are well-known in the art (see discussion inChapter 4 of “Advanced Organic Chemistry”, 4th edition J. March, JohnWiley and Sons, New York, 1992).

The compounds of Formula (I) may exhibit the phenomena of tautomerismand structural isomerism. For example, the compounds described hereinmay adopt an E or a Z configuration about the double bond connecting the2-indolinone moiety to the pyrrole moiety or they may be a mixture of Eand Z. This invention encompasses any tautomeric or structural isomericform and mixtures thereof which possess the ability to modulate RTK, CTKand/or STK activity and is not limited to any one tautomeric orstructural isomeric form.

A “pharmaceutical composition” refers to a mixture of one or more of thecompounds described herein, or physiologically/pharmaceuticallyacceptable salts or prodrugs thereof, with other chemical components,such as physiologically/pharmaceutically acceptable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

The compound of Formula (I) may also act as a prodrug. A “prodrug”refers to an agent which is converted into the parent drug in vivo.Prodrugs are often useful because, in some situations, they may beeasier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent drug is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the patent drug. An example, without limitation, of a prodrug wouldbe a compound of the present invention which is administered as an ester(the “prodrug”) to facilitate transmittal across a cell membrane wherewater solubility is detrimental to mobility but then is metabolicallyhydrolyzed to the carboxylic acid, the active entity, once inside thecell where water solubility is beneficial.

A further example of a prodrug might be a short polypeptide, forexample, without limitation, a 2-10 amino. acid polypeptide, bondedthrough a terminal amino group to a carboxy group of a compound of thisinvention wherein the polypeptide is hydrolyzed or metabolized in vivoto release the active molecule. The prodrugs of a compound of Formula(I) are within the scope of this invention.

Additionally, it is contemplated that a compound of Formula (I) would bemetabolized by enzymes in the body of the organism such as human beingto generate a metabolite that can modulate the activity of the proteinkinases. Such metabolites are within the scope of the present invention.

As used herein, a “physiologically/pharmaceutically acceptable carrier”refers to a carrier or diluent that does not cause significantirritation to an organism and does not abrogate the, biological activityand properties of the administered compound.

An “pharmaceutically acceptable excipient” refers to an inert substanceadded to a pharmaceutical composition to further facilitateadministration of a compound. Examples, without limitation, ofexcipients include calcium carbonate, calcium phosphate, various sugarsand types of starch, cellulose derivatives, gelatin, vegetable oils andpolyethylene glycols.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which retain the biological effectiveness and properties ofthe parent compound. Such salts include:

-   -   (i) acid addition salt which is obtained by reaction of the free        base of the parent compound with inorganic acids such as        hydrochloric acid, hydrobromic acid, nitric acid, phosphoric        acid, sulfuric acid, and perhcloric acid and the like, or with        organic acids such as acetic acid, oxalic acid, (D) or (L) malic        acid, maleic acid, methanesulfoniic acid, ethanesulfonic acid,        p-toluenesulfonic acid, salicylic acid, tartaric acid, citric        acid, succinic acid or malonic acid and the like, preferably        hydrochloric acid or (L)-malic acid such as the L-malate salt of        5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic        acid(2-diethylaminoethyl)amide; or    -   (2) salts formed when an acidic proton present in the parent        compound either is replaced by a metal ion, e.g., an alkali        metal ion, an alkaline earth ion, or an aluminum ion; or        coordinates with an organic base such as ethanolamine,        diethanolamine, triethanolamine, tromethamine,        N-methylglucamine, and the like.

“PK” refers to receptor protein tyrosine kinase (RTKs), non-receptor or“cellular” tyrosine kinase (CTKs) and serine-threonine kinases (STKs).

“Method” refers to manners, means, techniques and procedures foraccomplishing a given task including, but not limited to, those manners,means, techniques and procedures either known to, or readily developedfrom known manners, means, techniques and procedures by, practitionersof the chemical, pharmaceutical, biological, biochemical and medicalarts.

“Modulation” or “modulating” refers to the alteration of the catalyticactivity of RTKs, CTKs and STKs. In particular, modulating refers to theactivation of the catalytic activity of RTKS, CT Ks and STKs, preferablythe activation or inhibition of the catalytic activity of RTKs, CTKs andSTKs, depending on the concentration of the compound or salt to whichthe RTK, CTK or STK is exposed or, more preferably, the inhibition ofthe catalytic activity of RTKs, CTKs and STKS.

“Catalytic activity” refers to the rate of phosphorylation of tyrosineunder the influence, direct or indirect, of RTKs and/or CTKs or thephosphorylation of serine and threonine under the influence, direct orindirect, of STKs.

“Contacting” refers to bringing a compound of this invention and atarget PK together in such a manner that the compound can affect thecatalytic activity of the PK, either directly, i.e., by interacting withthe kinase itself, or indirectly, i.e., by interacting with anothermolecule on which the catalytic activity of the kinase is dependent.Such “contacting” can be accomplished “in vitro,” i.e., in a test tube,a petri dish or the like. In a test tube, contacting may involve only acompound and a PK of interest or it may involve whole cells. Cells mayalso be maintained or grown in cell culture dishes and contacted with acompound in that environment. In this context, the ability of aparticular compound to affect a PK related disorder, i.e., the IC₅₀ ofthe compound, defined below, can be determined before use of thecompounds in vivo with more complex living organisms is attempted. Forcells outside the organism, multiple methods exist, and are well-knownto those skilled in the art, to get the PKs in contact with thecompounds including, but not limited to, direct cell microinjection andnumerous transmembrane carrier techniques.

“In vitro” refers to procedures performed in an artificial environmentsuch as, e.g., without limitation, in a test tube or culture medium.

“In vivo” refers to procedures performed within a living organism suchas, without limitation, a mouse, rat or rabbit.

“PK related disorder,” “PK driven disorder,” and “abnormal PK activity”all refer to a condition characterized by inappropriate, i.e., under or,more commonly, over, PK catalytic activity, where the particular PK canbe an RTK, a CTK or an STK. Inappropriate catalytic activity can ariseas the result of either: (1) PK expression in cells which normally donot express PKs, (2) increased PK expression leading to unwanted cellproliferation, differentiation and/or growth, or, (3) decreased PKexpression leading to unwanted reductions in cell proliferation,differentiation and/or growth. Over-activity of a PK refers to eitheramplification of the gene encoding a particular PK or production of alevel of PK activity which can correlate with a cell proliferation,differentiation and/or growth disorder (that is, as the level of the PKincreases, the severity of one or more of the symptoms of the cellulardisorder increases). Under-activity is, of course, the converse, whereinthe severity of one or more symptoms of a cellular disorder increase asthe level of the PK activity decreases.

“Treat”, “treating” and “treatment” refer to a method of alleviating orabrogating a PK mediated cellular disorder and/or its attendantsymptoms. With regard particularly to cancer, these terms simply meanthat the life expectancy of an individual affected with a cancer will beincreased or that one or more of the symptoms of the disease will bereduced.

“Organism” refers to any living entity comprised of at least one cell. Aliving organism can be as simple as, for example, a single eukarioticcell or as complex as a mammal, including a human being.

“Therapeutically effective amount” refers to that amount of the compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disorder being treated. In reference to the treatment ofcancer, a therapeutically effective amount refers to that amount whichhas the effect of:

-   -   (1) reducing the size of the tumor;    -   (2) inhibiting (that is, slowing to some extent,preferably        stopping) tumor metastasis;    -   (3) inhibiting to some extent (that is, slowing to some extent,        preferably stopping) tumor growth, and/or,    -   (4) relieving to some extent (or, preferably, eliminating) one        or more symptoms associated with the cancer.

“Monitoring” means observing or detecting the effect of contacting acompound with a cell expressing a particular PK. The observed ordetected effect can be a change in cell phenotype, in the catalyticactivity of a PK or a change in the interaction of a PK with a naturalbinding partner. Techniques for observing or detecting such effects arewell-known in the art.

The above-referenced effect is selected from a change or an absence ofchange in a cell phenotype, a change or absence of change in thecatalytic activity of said protein kinase or a change or absence ofchange in the interaction of said protein kinase with a natural bindingpartner in a final aspect of this invention.

“Cell phenotype” refers to the outward appearance of a cell or tissue orthe biological function of the cell or tissue. Examples, withoutlimitation, of a cell phenotype are cell size, cell growth, cellproliferation, cell differentiation, cell survival, apoptosis, andnutrient uptake and use. Such phenotypic characteristics are measurableby techniques well-known in the art.

“Natural binding partner” refers to a polypeptide that binds to aparticular PK in a cell. Natural binding partners can play a role inpropagating a signal in a PK-mediated signal transduction process. Achange in the interaction of the natural binding partner with the PK canmanifest itself as an increased or decreased concentration of thePK/natural binding partner complex and, as a result, in an observablechange in the ability of the PK to mediate signal transduction.

Representative compounds of the present invention are shown in Table Ibelow. TABLE 1 Exam- ple Structure Name  1

4-Methyl-5-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid  2

4-Methyl-5-(1-methyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2 carboxylic acid  3

4-Methyl-5-(2-oxo-1,2-dihydroindol- 3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid methyl ester  4

5-(5-Chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-methyl-1H-pyrrole-2 carboxylic acid ethyl ester  5

5-(5-Chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-methyl-1H-pyrrole-2 carboxylic acid  6

5-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-methyl-1H-pyrrole-2 carboxylic acid(3-pyrrolidin-1-ylpropyl)amide  7

5-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-methyl-1H-pyrrole-2 carboxylic acid(3-diethylaminopropyl)amide  8

5-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (2-diethylaminoethyl)amide 9

5-(2-Oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (2-diethylaminoethyl)amide 10

5-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid(2-diethylaminoethyl)methylamide  11

5-(2-Oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid(2-diethylaminoethyl)methylamide  12

3-Methyl-5-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (3-diethylaminopropyl)amide 13

5-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2 carboxylic acid(3-diethylaminopropyl)amide  14

3-Methyl-5-(2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2 carboxylic acid(3-diethylaminopropyl)amide  15

5-(5-Methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole 2-carboxylic acid(3-diethylaminopropyl)amide  16

5-(6-Methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole 2-carboxylic acid(3-diethylaminopropyl)amide  17

3-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1- carboxylic acid(2-diethylaminoethyl)amide  18

3-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole- 1-carboxylic acid(3-diethylaminopropyl)amide  19

3-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole- 1-carboxylic acid(3-pyrrolidin-1-ylpropyl)amide  20

3-(2-Oxo-6-pyridin-3-yl-1,2-dihydroindol-3- ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid (2-diethylaminoethyl)amide  21

4-Benzoyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2- carboxylic acid(3-diethylaminopropyl)amide  22

4-Benzoyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole- 2-carboxylic acid(3-morpholin-4-ylpropyl)amide  23

4-Benzoyl-3-methyl-5-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole- 2-carboxylic acid(3-pyrrolidin-1-ylpropyl)amide  24

4-Benzoyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole- 2-carboxylic acid(3-pyrrolidin-1-ylpropyl)amide  25

4-Benzoyl-3-methyl-5-(2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2- carboxylic acid(3-pyrrolidin-1-ylpropyl)amide  26

4-Benzoyl-5-(6-methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole- 2-carboxylic acid(3-pyrrolidin-1-ylpropyl)amide  27

4-Benzoyl-5-(5-methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole- 2-carboxylic acid(3-pyrrolidin-1-ylpropyl)amide  28

4-Benzoyl-5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole- 2-carboxylic acid(3-pyrrolidin-1-ylpropyl)amide  29

4-Acetyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole- 2-carboxylic acid(3-diethylaminopropyl)amide  30

4-Acetyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole- 2-carboxylic acid(3-pyrrolidin-1-ylpropyl)amide  31

4-Acetyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole- 2-carboxylic acid(3-morpholin-4-ylpropyl)amide  32

4-Acetyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole- 2-carboxylic acid(3-hydroxy-propyl)amide  33

4-Acetyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole- 2-carboxylic acid(2-hydroxy-ethyl)amide  34

4-Acetyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole- 2-carboxylic acid(2-morpholin-4-yl-ethyl)amide  35

4-Acetyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole- 2-carboxylic acid(2-pyrrolidin-1-yl-ethyl)amide  36

4-Acetyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole- 2-carboxylic acid[2-(4-hydroxy-phenyl)-ethyl]amide  37

5-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-isopropyl-4-phenyl-1H-pyrrole- 3-carboxylic acid(3-diethylaminopropyl)amide  38

5-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-isopropyl-4-phenyl-1H-pyrrole- 3-carboxylic acid(3-pyrrolidin-1-ylpropyl)amide  39

5-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-isopropyl-4-phenyl-1H-pyrrole- 3-carboxylic acid(2-diethylaminoethyl)amide  40

5-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-isopropyl-4-phenyl-1H-pyrrole- 3-carboxylic acid[3-(4-methyl-piperazin-1-yl)- propyl]amide  41

5-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-isopropyl-4-phenyl-1H-pyrrole- 3-carboxylic acid  42

5-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-methyl-4-phenyl-1H-pyrrole- 3-carboxylic acid(2-pyrrolidin-1-yl-ethyl)amide  43

5-[6-(2-Methoxy-phenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2-methyl- 4-phenyl-1H-pyrrole-3-carboxylicacid (2- pyrrolidin-1-yl-ethyl)amide  44

5-(5-Bromo-2-oxo-1,2-dihydroindol-3- ylidenemethyl)-2-methyl-4-phenyl-1Hpyrrole-3-carboxylic acid (2-dimethylamino- ethyl)amide  45

5-[6-(2-Methoxy-phenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2-methyl-4- phenyl-1H-pyrrole-3-carboxylicacid (2- dimethylamino-ethyl)amide  46

5-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-methyl-4-phenyl-1H- pyrrole-3-carboxylic acid ethylester  47

5-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-methyl-4-phenyl-1H- pyrrole-3-carboxylic acid(3-diethylaminopropyl) amide  48

5-(5-Bromo-2-oxo-1,2-dihydroindol-3- ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-dimethylamino-ethyl) amide  49

2,4-Dimethyl-5-(2-oxo-6-phenyl-1,2- dihydroindol-3-yldenemethyl)-1H-pyrrole-3-carboxylic acid (2-dimethylamino-ethyl) amide  50

5-(5-Chloro-2-oxo-1,2-dihydroindol-3- ylidenemethyl)-2,4-methyl-1H-pyrrole-3-carboxylic acid (2-dimethylamino-ethyl) amide  51

5-(5-Bromo-2-oxo-1,2-dihydroindol-3- ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl) amide  52

5-(5-Bromo-2-oxo-1,2-dihydroindol-3- ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-yl-ethyl) amide  53

5-(5-Bromo-2-oxo-1,2-dihydroindol-3- ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (3-imidazol-1-ylpropyl) amide  54

5-[6-(2-Methoxy-phenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2,4-dimethyl- 1H-pyrrole-3-carboxylic acid(2- dimethylamino-ethyl)amide  55

5-[6-(3-Methoxy-phenyl)-2-oxo-1,2- dihydroindol-3-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-dimethylamino-ethyl)amide  56

2,4-Dimethyl-5-(2-oxo-5-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole- 3-carboxylic acid(2-diethylaminoethyl)amide  57

2,4-Dimethyl-5-(2-oxo-5-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole- 3-carboxylic acid(2-pyrrolidin-1-yl-ethyl)amide  58

2,4-Dimethyl-5-(2-oxo-5-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole- 3-carboxylic acid(3-imidazol-1-ylpropyl)amide  59

2,4-Dimethyl-5-(2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3- carboxylic acid(2-diethylaminoethyl)amide  60

2,4-Dimethyl-5-(2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3- carboxylic acid(2-pyrrolidin-1-yl-ethyl)amide  61

2,4-Dimethyl-5-(2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3- carboxylic acid(3-imidazol-1-ylpropyl)amide  62

5-[6-(3,5-Dichloro-phenyl)-2-oxo-1,2-dihydroindol-3-yldenemethyl]-2,4-dimethyl-1H- pyrrole-3-carboxylic acid(2- diethylaminoethyl)amide  63

2,4-Dimethyl-5-(2-oxo-6-pyridin-3-yl-1,2-dihydroindol-3-ylidenemethyl)-1H- pyrrole-3-carboxylic acid(2-diethylaminoethyl) amide  64

2,4-Dimethyl-5-(2-oxo-6-pyridin-3-yl-1,2-dihydroindol-3-ylidenemethyl)-1H- pyrrole-3-carboxylic acid(2-pyrrolidin-1-yl- ethyl)amide  65

2,4-Dimethyl-5-(2-oxo-6-pyridin-3-yl-1,2-dihydroindol-3-ylidenemethyl)-1H- pyrrole-3-carboxylic acid(3-dimethylamino- propyl)amide  66

2,4-Dimethyl-5-(2-oxo-5-phenyl-1,2- dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic acid (3-dimethylamino- propyl)amide  67

2,4-Dimethyl-5-(2-oxo-5-phenyl-1,2- dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic acid (3-diethylaminopropyl) amide  68

2,4-Dimethyl-5-(2-oxo-6-phenyl-1,2- dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic acid (3-diethylaminopropyl) amide  69

3-[4-(3-Diethylamino-propylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-ylmethylene)- 2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid (3-chloro-4-methoxy- phenyl)amide  70

5-(5-Bromo-2-oxo-1,2-dihydroindol-3- ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (3-diethylaminopropyl) amide  71

5-(5-Bromo-2-oxo-1,2-dihydroindol-3- ylidenemethyl)-2,4-diisopropyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl) amide  72

5-(5-Bromo-2-oxo-1,2-dihydroindol-3- ylidenemethyl)-2,4-diisopropyl-1H-pyrrole-3-carboxylic acid (3-diethylaminopropyl) amide  73

5-(5-Bromo-2-oxo-1,2-dihydroindol-3- ylidenemethyl)-2,4-diisopropyl-1H-pyrrole-3-carboxylic acid (3-pyrrolidin-1- ylpropyl)amide  74

5-(5-Bromo-2-oxo-1,2-dihydroindol-3- ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (pyridin-4-ylmethyl) amide  75

5-[6-(4-Butyl-phenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2,4-dimethyl- 1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-yl- ethyl)amide  76

5-[6-(5-isopropyl-2-methoxy-phenyl)-2-oxo-1,2- dihydroindol-3-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-pyrrolidin-1-yl- ethyl)amide  77

5-[6-(4-Ethyl-phenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2,4-dimethyl- 1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1- yl-ethyl)amide  78

5-[6-(2,4-Dimethoxy-phenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2,4- dimethyl-1H-pyrrole-3-carboxylic acid(2- pyrrolidin-1-yl-ethyl)amide  79

5-[6-(3-Isopropyl-phenyl)-2-oxo-1,2- dihydroindol-3-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2- pyrrolidin-1-yl-ethyl)amide 80

5-(5-Fluoro-2-oxo-1,2-dihydroindol-3- ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl) amide  81

3-[4-(2-diethylaminoethylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-ylmethylene]2-oxo-2,3-dihydro-1H-indole-6-carboxylicacid  82

5-(5-Dimethylsulfamoyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole- 3-carboxylic acid(2-pyrrolidin-1-yl-ethyl)amide  83

5-[5-(3-Chloro-phenylsulfamoyl-2-oxo-1,2-dihydrolindol-3-ylidenemethyl]-2,4- dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-yl-ethyl)amide  84

2,4-Dimethyl-5-[2-oxo-5-(pyridin-3- ylsulfamoyl)-1,2-dihydroindol-3-ylidenemethyl]-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-yl-ethyl)amide  85

3-[3,5-Dimethyl-4-(4-methyl-piperazine-1- carbonyl)-1H-pyrrol-2-ylmethylene]-4-(2-hydroxy-ethyl)-1,3- dihydroindol-2-one  86

3-[3,5-Dimethyl-4-(4-methyl-piperazine-1- carbonyl)-1H-pyrrol-2-ylmethylene]-2-oxo-2,3-dihydro-1H- indole-5-sulfonic acid phenylamide 87

5-(5-Dimethylsulfamoyl-2-oxo-1,2- dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide  88

5-[5-(3-Chloro-phenylsulfamoyl]-2-oxo-1,2-dihydroindol-3-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3- carboxylicacid (2-diethylaminoethyl)amide  89

3-(5-Bromo-2-oxo-1,2-dihydro-indol-3- ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid (2-dimethylamino-ethyl)-amide  90

3-(2-Oxo-1,2-dihydro-indol-3-ylidenemethyl)- 4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid ethyl ester  91

3-(4-Methyl-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-4,5,6,7-tetrahydro- 2H-isoindole-1-carboxylic acid ethylester  92

3-(5-Bromo-2-oxo-1,2-dihydro-indol-3- ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid ethyl ester  93

3-(3-Ethoxycarbonyl-4,5,6,7-tetrahydro-2H-isoindol-1-ylmethylene)-2-oxo- 2,3-dihydro-1H-indole-5-carboxylic acid 94

3-(5-Methoxy-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-4,5,6,7-tetrahydro- 2H-isoindole-1-carboxylic acid ethylester  95

3-(2-Oxo-5-phenyl-1,2-dihydro-indol-3-ylidenemethyl)-4,5,6,7-tetrahydro- 2H-isoindole-1-carboxylic acid ethylester  96

3-(2-Oxo-5-sulfamoyl-1,2-dihydro-indol-3-ylidenemethyl)-4,5,6,7-tetrahydro- 2H-isoindole-1-carboxylic acid ethylester  97

3-(5-Methylsulfamoyl-2-oxo-1,2-dihydro-indol- 3-ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid ethyl ester  98

3-(5-Dimethylsulfamoyl-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-4,5,6,7- tetrahydro-2H-isoindole-1-carboxylic acidethyl ester  99

3-(2-Oxo-5-phenylsulfamoyl-1,2-dihydro-indol-3- ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid ethyl ester 100

3-(6-Bromo-2-oxo-1,2-dihydro-indol-3- ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid ethyl ester 101

3-(2-Oxo-6-phenyl-1,2-dihydro-indol-3-ylidenemethyl)-4,5,6,7-tetrahydro- 2H-isoindole-1-carboxylic acid ethylester 102

3-(3-Ethoxycarbonyl-4,5,6,7-tetrahydro-2H-isoindol-1-ylmethylene)-2-oxo- 2,3-dihydro-1H-indole-6-carboxylic acid103

3-(6-Methoxy-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-4,5,6,7-tetrahydro- 2H-isoindole-1-carboxylic acid ethylester 104

3-(5-isopropylsulfamoyl-2-oxo-1,2-dihydro-indol-3-ylidenemethyl]-4,5,6,7- tetrahydro-2H-isoindole-1-carboxylicacid ethyl ester 105

3-(3-Methylcarbamoyl-4,5,6,7-tetrahydro-2H- isoindol-1-ylmethylene)-2-oxo-2,3-dihydro-1H-indole-5-carboxylic acid 106

3-(3-Dimethylcarbamoyl-4,5,6,7-tetrahydro-2H- isoindol-1-ylmethylene)-2-oxo-2,3-dihydro-1H-indole-5-carboxylic acid 107

2-Oxo-3-[3-pyrrolidine-1-carbonyl-4,5,6,7- tetrahydro-2H-isoindol-1-ylmethylene]-2,3-dihydro-1H-indole-5- carboxylic acid 108

3-[3-(Morpholine-4-carbonyl)-4,5,6,7- tetrahydro-2H-isoindol-1-ylmethylene]-2-oxo-2,3-dihydro-1H-indole-5- carboxylic acid 109

3-[3-(Morpholine-4-carbonyl)-4,5,6,7- tetrahydro-2H-isoindol-1-ylmethylene]-2-oxo-2,3-dihydro-1H-indole-6- carboxylic acid 110

3-(5-Bromo-2-oxo-1,2-dihydro-indol-3- ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid methylamide 111

3-(5-Bromo-2-oxo-1,2-dihydro-indol-3- ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid dimethylamide 112

5-Bromo-3-[3-(pyrrolidine-1-carbonyl)-4,5,6,7-tetrahydro-2H-isoindole-1- ylmethylene]-1,3-dihydro-indol-2-one 113

5-Bromo-3-[3-(morpholine-4-carbonyl)-4,5,6,7- tetrahydro-2H-isoindole-1-ylmethylene]-1,3-dihydro-indol-2-one 114

3-(3-Dimethylcarbamoyl-4,5,6,7-tetrahydro-2H- isoindol-1-ylmethylene)-2-oxo-2,3-dihydro-1H-indole-6-carboxylic acid 115

4-Methyl-5-(5-methylsulfamoyl-2-oxo-1,2- dihydro-indol-3-yldenemethyl)-1H-pyrrole-3-carboxylic acid 116

{[4-Methyl-5-(4-methyl-5-methylsulfamoyl-2- oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carbonyl]amino}- acetic acid ethyl ester 117

{[4-Methyl-5-(5-methylsulfamoyl-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)- 1H-pyrrole-3-carbonyl]-amino}- aceticacid ethyl ester 118

{[4-Methyl-5-(5-methylsulfamoyl-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)- 1H-pyrrole-3-carbonyl]-amino}-aceticacid 119

3-[3-Methyl-4-(piperidine-1-carbonyl)-1H-pyrrol-2-ylmethylene]-2-oxo-2,3- dihydro-1H-indole-5-sulfonic acid methylamide120

5-Methyl-2-(2-oxo-1,2-dihydro-indol-3- ylidenemethyl)-1H-pyrrole-3-carboxylic acid 121

5-Methyl-2-(2-oxo-1,2-dihydro-indol-3- ylidenemethyl)-1H-pyrrole-3-carboxylic acid ethyl ester 122

2-(5-Bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-5-methyl-1H-pyrrole- 3-carboxylic acid ethyl ester 123

2-(5-Bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-5-methyl-1H-pyrrole- 3-carboxylic acid 124

2-(5-Bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-5-methyl-1H-pyrrole- 3-carboxylic acid(2-pyrrolidin-1-yl-ethyl)-amide 125

2-(5-Bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-5-methyl-1H-pyrrole- 3-carboxylic acid(2-diethylamino-ethyl)-amide 133

5-[5-Chloro-2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole- 3-carboxylic acid(2-acetylamino-ethyl)- amide 399 [M − 1] 134

5-[5-Fluoro-2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole- 3-carboxylic acid(2-acetylamino-ethyl)- amide 383 [M − 1] 135

2,4-Dimethyl-5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-1H-pyrrole-3- carboxylic acid (2-acetylamino-ethyl)-amide 365 [M − 1] 136

5-[5-Bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole- 3-carboxylic acid[3-(2-oxo-tetrahydro- pyrimidin-1-yl)-propyl]-amide 500 [M + 1]502 [M +1] 137

5-[5-Chloro-2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole- 3-carboxylic acid[3-(2-oxo-tetrahydro- pyrimidin-1-yl)-propyl]-amide 454 [M − 1] 138

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole- 3-carboxylic acid[3-(2-oxo-tetrahydro- pyrimidin-1-yl)-propyl]-amide 438 [M − 1] 139

2,4-Dimethyl-5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-1H-pyrrole-3- carboxylic acid[3-(2-oxo-tetrahydro-pyrimidin- 1-yl)-propyl]-amide 422 [M + 1] 140

5-[5-Cyano-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole- 3-carboxylic acid[3-(2-oxo-tetrahydro- pyrimidin-1-yl)-propyl]-amide 447 [M + 1] 141

Trifluoro-acetate4-[2-({5-[5- bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4dimethly-1H-pyrrole-3-carbonyl}-amino)- ethyl]-2-oxo-piperazin-1-ium;486 [M + 1]488 [M + 1] 126

2,4-Dimethyl-5-[2-oxo-1,2- dihydro- indol-(3Z)-ylidenemethyl]-1H-pyrrole-3-carboxylic acid (2- diethylaminoethyl)-amide 381 [M + 1]127

5-[5-Chloro-2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)-amide 415[M + 1] 128

2,4-Dimethyl-5-[2-oxo-1,2- dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid (2-pyrrolidin-1-ylethyl)-amide 379 [M + 1] 129

5-[5-Fluoro-2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-ylethyl)-amide 397[M + 1] 130

5-[5-Chloro-2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-1H-pyrrole-3-carboxylic acid (2- pyrrolidin-1-ylethyl)-amide 413 [M + 1]131

2,4-Dimethyl-5-[2-oxo-1,2- dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid (2- dimethylaminoethyl)-amide353 [M + 1] 132

5-[5-Fluoro-2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-dimethylaminoethyl)-amide 371[M + 1] 142

5-[5-Cyano-2-oxo-1,2-dihydro-indol- (3Z)-ylidenemethyl]-2,4-dimethyl-1Hpyrrole-3-carboxylic acid [3-(2-oxo- pyrrolidin-1-yl)-propyl]-amide 430[M − 1] 143

5-[5-Bromo-2-oxo-1,2-dihydro-indol- (3Z)-ylidenemethyl]-2,4-dimethyl-1Hpyrrole-3-carboxylic acid [2-(2-oxo- imidazolidin-1-yl)-ethyl]-amide 470[M − 1]472 [M − 1] 144

5-[5-Chloro-2-oxo-1,2-dihydro-indol- (3Z)-ylidenemethyl]-2,4-dimethyl-1Hpyrrole-3-carboxylic acid [2-(2-oxo- imidazolidin-1-yl)-ethyl]-amide 428[M + 1] 145

5-[5-Fluoro-2-oxo-1,2-dihydro-indol- (3Z)-ylidenemethyl]-2,4-dimethyl-1Hpyrrole-3-carboxylic acid]2-(2-oxo- imidazolidin-1-yl)-ethyl]-amide 412[M + 1] 146

2,4-Dimethyl-5-[2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-1H-pyrrole3-carboxylic acid]2-(2-oxo- imidazolidin-1-yl)-ethyl]amide 392 [M − 1]147

5-[5-Cyano-2-oxo-1,2-dihydro-indol- (3Z)-ylidenemethyl]-2,4-dimethyl-1Hpyrrole-3-carboxylic acid]2-(2-oxo- imidazolin-1-yl)-ethyl]amide 419[M + 1] 148

{4-[2-({5-[5-Bromo-2-oxo-1,2- dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}amino)-ethyl]-piperazin-1-yl}-aceticacid ethyl ester 558 [M + 1]560 [M + 1] 149

{4-[2-({5-[5-Chloro-2-oxo-1,2- dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}amino)-ethyl]-piperazin-1-yl}-aceticacid ethyl ester 514 [M + 1] 150

{4-[2-({5-[5-Fluoro-2-oxo-1,2- dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}- amino)-ethyl]-piperazin-1-yl}-aceticacid ethyl ester 498 [M + 1] 153

2,4-Dimethyl-5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-1H-pyrrole-3- carboxylic acid[2-(cyanomethyl-amino)- ethyl]-amide 362 [M − 1] 154

5-[5-Bromo-2-oxo-1,2-dihydro-indol- (3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3 carboxylic acid[3-(2-oxo-azepan-1-yl)-propyl]- amide 511 [M − 1]513 [M − 1] 155

5-[5-Chloro-2-oxo-1,2-dihydro-indol- (3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3 carboxylic acid[3-(2-oxo-azepan-1-yl)-propyl]- amide 469 [M + 1] 156

5-[5-Fluoro-2-oxo-1,2-dihydro-indol- (3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3 carboxylic acid[3-(2-oxo-azepan-1-yl)- propyl]-amide 453 [M + 1] 157

2,4-Dimethyl-5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-1H-pyrrole-3- carboxylic acid[3-(2-oxo-azepan-1-yl)- propyl]-amide 435 [M + 1] 158

5-[5-Cyano-2-oxo-1,2-dihydro-indol- (3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3 carboxylic acid[3-(2-oxo-azepan-1-yl)- propyl]-amide 460 [M + 1] 159

5-[5-Bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3 carboxylic acid(2-acetylamino-ethyl)- amide 443 [M − 1]445 [M − 1] 160

Trifluoro-acetate4-[2-({5-[5-fluoro-2- oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethly-1H- pyrrole-3-carbonyl}-amino)-ethyl]-2-oxo-piperazin-1-ium; 426 [M + 1] 161

Trifluoro-acetate4-[2-({2,4-dimethyl-5- [2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-1H-pyrrole-3-carbonyl}-amino)-ethyl]-2-oxo-piperazin-1-ium; 408 [M + 1] 162

Trifluoro-acetate4-[2-({5-[5-cyano-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}-amino)-ethyl]-2-oxo-piperazin-1-ium; 433 [M + 1]488 [M + 1] 163

5-[5-Bromo-2-oxo-1,2-dihydro-indol- (3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2-(2-cyano- ethylamino)-ethyl]-amide 454 [M −1]456 [M − 1] 164

5-[5-Chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H- pyrrole-3-carboxylic acid[2-(2-cyano- ethylamino)-ethyl]-amide 410 [M − 1] 165

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H- pyrrole-3-carboxylic acid[2-(2-cyano- ethylamino)-ethyl]-amide 394 [M − 1] 166

2,4-Dimethyl-5-[2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-1H-pyrrole-3 carboxylic acid [2-(2-cyano- ethylamino)-ethyl]-amide 376 [M −1] 167

5-[5-Cyano-2-oxo-1,2-dihydro-indol- (3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2-(2-cyano- ethylamino)-ethyl]-amide 401 [M −1] 168

Trifluoro-acetate4-[2-({5-[5-chloro-2- oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H- pyrrole-3-carbonyl}-amino)-ethyl]-2-oxo-piperazin-1-ium; 440 [M − 1] 168

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid[2-(4-methyl-piperazin-1-yl)- ethyl]-amide 424 [M − 1] 169

5-[5-Chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid[2-(4-methyl-piperazin-1-yl)- ethyl]-amide 440 [M − 1] 170

5-[5-Bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid[2-(4-methyl-piperazin-1-yl)- ethyl]-amide 484 [M − 1]486 [M − 1] 171

2,4-Dimethyl-5-[2-oxo-1,2-dihydro-indol-(3Z)ylidenemethyl]-1H-pyrrole-3-carboxylic acid[2-(4-methyl-piperazin-1-yl)-ethyl]- amide 406 [M − 1] 172

2,4-Dimethyl-5-[2-oxo-1,2-dihydro-indol-(3Z)ylidenemethyl]-1H-pyrrole-3-carboxylic acid[2-(3,5-dimethyl-piperazin-1-yl)-ethyl]- amide 422 [M + 1] 173

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid[2-(3,5-dimethyl-piperazin-1 yl)-ethyl]-amide 438 [M − 1] 174

5-[5-Chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid[2-(3,5-dimethyl-piperazin-1 yl)-ethyl]-amide 456 [M + 1] 175

5-[5-Bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid[2-(3,5-dimethyl-piperazin-1 yl)-ethyl]-amide 498 [M − 1]500 [M − 1] 176

2,4-Dimethyl-5-[2-oxo-1,2- dihydro-indol-(3Z)-ylidenemethyl]-1H-pyrrole-3-carboxylic acid [3- (4-methyl-piperazin-1-yl)-propyl]-amide 422 [M + 1] 177

5-[5-Fluoro-2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid [3-(4-methyl-piperazin-1-yl)-propyl]-amide 438 [M − 1] 178

5-[5-Chloro-2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid [3-(4-methyl-piperazin-1-yl)-propyl]-amide 454 [M − 1] 179

5-[5-Bromo-2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid [3-(4-methyl-piperazin-1-yl)-propyl]-amide 498 [M − 1]500 [M − 1] 180

2,4-Dimethyl-5-[2-oxo-1,2- dihydro-indol-(3Z)-ylidenemethyl]-1H-pyrrole-3-carboxylic acid [2- (4-benzyl-piperazin-1-yl)-ethyl]- amide482 [M − 1] 181

5-[5-Fluoro-2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2-(4-benzyl-piperazin-1-yl)-ethyl]-amide 500 [M − 1] 182

5-[5-Chloro-2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2-(4-benzyl-piperazin-1-yl)-ethyl]-amide 517 [M − 1] 183

5-[5-Bromo-2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2-(4-benzyl-piperazin-1-yl)-ethyl]amide 560 [M − 1]562 [M − 1] 184

5-[5-Chloro-2-oxo-1,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid [3-pyrrolidin-1yl-2-one)- amide480 [M + 1] 185

Trifluoro-acetate 4-[2-({5-[5- Chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}-amino)-ethyl]2-oxo-piperazin-1-ium 440[M − 1] 186

5-[5-Chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid[3-pyrrolidin-1yl-2-one)- amide 187

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(3-pyrrolidin-1yl-2-one)- amide 188

5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(3-pyrrolidin-1yl-2-one)- amide 189

5-[5-Chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-pyridin-2-ylethyl)-amide 190

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-pyridin-2-ylethyl)-amide trifluororacetate salt 191

5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-pyridin-2-ylethyl)-amide hydrochloride salt 192

5-[5-Bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-pyridin-2-ylethyl)-amide trifluororacetate salt 193

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-ethylaminoethyl)-amide 194

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-aminoethyl)-amide 195

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-2,4-dimethyl- 1H-pyrrole-3-carboxylic acid(2-diethyl-N- oxoaminoethyl)-amide 196

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-ethyl-N-hydroxy- aminoethyl)-amide 197

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-1H-pyrrole-3-carboxylic acid(2-diethylamino-2-hydroxyethyl)- amide 198

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H- pyrrole-3-carboxylic acid [2-ethyl-2-(2-hydroxyethyl)aminoethyl]-amide 199

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid [2-ethyl-2-(1-hydroxyethyl)aminoethyl]-amide 200

5-[5-Cyano-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-N-acetylaminoethyl)- amide 201

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(carboxymethyl)-amide 202

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)- ylidenemethyl]-1H-pyrrole-3-carboxylic acid [2-(2-hydroxethylamino)ethyl]- amide 203

5-[5-Cyano-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-1H-pyrrole-3-carboxylic acid (2-pyridin-2-ylethyl)-amidetrifluoroacetate 204

5-[5-Bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-1H-pyrrole-3-carboxylic acid(3-pyrrolidin-1-yl-2-onepropyl)-amide trifluoroacetate

The compound numbers correspond to the Example numbers in the Examplessection. That is, the synthesis of Compound 1 in Table 1 is described inExample 1. The compounds presented in Table 1 are exemplary only and arenot to be construed as limiting the scope of this invention in anymanner.

PREFERRED EMBODIMENTS

While the broadest definition is set forth in the Summary of theInvention, certain compounds of Formula (I) set forth below arepreferred.

(1) A preferred group of compounds of Formula (I) is that wherein R¹,R³, and R⁴ are hydrogen.

(2) Another preferred group of compounds of Formula (I) is that whereinR¹, R², and R⁴ are hydrogen.

(3) Another preferred group of compounds of Formula (I) is that whereinR¹, R², and R³ are hydrogen.

(4) Another preferred group of compounds of Formula (I) is that whereinR², R³, and R⁴ are hydrogen.

(5) Another preferred group of compounds of Formula (I) is that whereinR¹, R², R³ and R⁴ are hydrogen.

(6) Yet another preferred group of compounds of Formula (I) is thatwherein R⁵, R⁶ or R⁷, preferably R⁵ or R⁶, more preferably R⁶ is —COR¹⁰wherein R¹⁰ is —NR¹¹(CH₂)_(n)R¹² wherein:

-   -   R¹¹ is hydrogen or lower unsubstituted alkyl, preferably        hydrogen or methyl;    -   n is 2, 3 or 4, preferably 2 or 3; and    -   R¹² is —NR¹³R¹⁴ wherein R¹³ and R¹⁴ are independently alkyl,        more preferably lower unsubstituted lower alkyl or R¹³ and R¹⁴        combine to form a group selected from —(CH₂)₄—, —(CH₂)₅—,        —(CH₂)₂—O—(CH₂)₂— or —(CH₂)₂N(CH₃)(CH₂)₂—, preferably R¹³ and        R¹⁴ are independently hydrogen, methyl, ethyl, or combine to        form morpholin-4-yl, pyrrolidin-1-yl, piperazin-1-yl, or        4-methylpiperazin-1-yl.

More preferably, R⁵ or R⁶ in (6) above isN-(2-dimethylaminoethyl-)aminocarbonyl,N-(2-ethylaminoethyl)-N-methylaminocarbonyl,N-(3-dimethylaminopropyl)-aminocarbonyl,N-(2-diethylaminoethyl)aminocarbonyl,N-(3-ethylaminopropyl)aminocarbonyl,N-(3-diethylaminopropyl)aminocarbonyl,3-pyrrolidin-1-yl-propylaminocarbonyl,3-morpholin-4-ylpropyl-aminocarbonyl,2-pyrrolidin-1-ylethylaminocarbonyl, 2-morpholin-4-ylethylaminocarbonyl,2-(4-methylpiperazin-1-yl)ethylaminocarbonyl,2-(4-methylpiperazin-1-yl)propylaminocarbonyl,2-(3,5-dimethylpiperazin-1-y)ethylaminocarbonyl or2-(3,5-dimethylpiperazin-1-y)propylaminocarbonyl, even more preferablyN-(2-diethyl-aminoethyl)aminocarbonyl orN-(2-ethylaminoethyl)amino-carbonyl.

(7) Yet another preferred group of compounds of Formula (I) is thatwherein R⁵, R⁶ or R⁷, preferably R⁵ or R⁶, more preferably R⁶ is —COR¹⁰wherein R¹⁰ is —NR³R¹⁴ wherein R¹³ is hydrogen and R¹⁴ is alkyl,preferably lower alkyl substituted with hydroxy, aryl, heteroaryl,heteroalicyclic, or carboxy, more preferably methyl, ethyl, propyl orbutyl substituted with hydroxy, aryl, heteroalicyclic such aspiperidine, piperazine, morpholine and the like, heteroaryl, or carboxy.Even more preferably within this group (7), R⁵ or R⁶ is2-ethoxycarbonylmethyl-aminocarbonyl, carboxymethylamino-carbonyl,3-hydroxypropyl-aminocarbonyl, 2-hydroxyethylaminocarbonyl,3-triazin-1-ylpropylamino-carbonyl, triazin-1-ylethylaminocarbonyl,4-hydroxy-phenylethylaminocarbonyl, 3-imidazol-1-ylpropyl-aminocarbonyl,pyridin-4-ylmethylaminocarbonyl, 2-pyridin-2-ylethylaminocarbonyl or2-imidazol-1-ylethylaminocarbonyl.

(8) Yet another preferred group of compounds of Formula (I) is thatwherein R⁵, R⁶ or R⁷, preferably R⁵ or R⁶, more preferably R⁶ is —COR¹⁰wherein R¹⁰ is —NR¹¹(CH₂)_(n)R¹² wherein:

-   -   R¹¹ is hydrogen or alkyl, preferably hydrogen or methyl;    -   n is 2, 3 or 4, preferably 2 or 3; and    -   R¹² is —NR¹³R¹⁴ wherein R¹³ and R¹⁴ together combine to form a        heterocycle, preferably a 5, 6 or 7 membered heterocycle        containing a carbonyl group and 1 or 2 nitrogen atoms.        Preferably, R⁵ or R⁶ is        2-(3-ethoxycarbonylmethylpiperazin-1-yl)ethylaminocarbonyl,        2-(3-oxopiperazin-1-yl)ethylaminocarbonyl,        2-(imidazolidin-1-yl-2-one)ethylaminocarbonyl,        2-(tetrahydropyrimidin-1-yl-2-one)ethylaminocarbonyl,        2-(2-oxopyrrolidin-1-yl)-ethylaminocarbonyl,        3-(4-methylpiperazin-1-yl)-propylaminocarbonyl,        3-(3-ethoxycarbonylmethylpiperazin-1-yl)-propylaminocarbonyl,        3-(3-oxopiperazin-1-yl)propyl-aminocarbonyl,        3-(imidazolidin-1-yl-2-one)propyl-aminocarbonyl,        3-(tetrahydropyrimidin-1-yl-2-one)-propylaminocarbonyl,        3-(2-oxopyrrolidin-1-yl)propyl-aminocarbonyl,        2-(2-oxohomopiperidin-1-yl)ethylamino-carbonyl or        3-(2-oxohomopiperidin-1-yl)propylaminocarbonyl.

(9) Yet another preferred group of compounds of Formula (I) is thatwherein R⁵, R⁶ or R⁷, preferably R⁵ or R⁶, more preferably, R⁶ is —COR¹⁰wherein:

-   -   (a) R¹⁰ is —NR¹¹(CH₂)_(n)R¹² wherein:        -   R¹¹ is hydrogen or alkyl, preferably hydrogen or methyl;        -   n is 2, 3 or 4, preferably 2 or 3; and        -   R¹² is —NR¹³R¹⁴ wherein R¹³ is hydrogen and R¹⁴ is            cyanoalkyl or —NHCOR^(a) where R^(a) is alkyl; or    -   (b) R¹⁰ is —NR¹³R¹⁴ wherein R¹³ and R¹⁴ together combine to form        a heterocycle not containing a carbonyl group within the ring.        Preferably, R⁵ or R⁶ is 2-(2-cyanoethylamino)ethylaminocarbonyl,        2-(acetylamino)-ethylaminocarbonyl, morpholinocarbonyl,        piperidin-1-yl-carbonyl, 2-cyanomethylaminoethylaminocarbonyl or        piperidin-1-ylcarbonyl.

(10) Another preferred group of compouds of Formula (I) is that whereinR⁵ is —COR¹⁰ wherein R¹⁰ is —NR¹³R¹⁴ wherein R¹³ is hydrogen and R¹⁴ islower alkyl substituted with hydroxy, lower alkyl substituted withhydroxyalkylamino, carboxy, or —NR¹⁸R¹⁹ wherein R¹⁸ and R¹⁹ areindependently hydrogen or lower unsubstituted alkyl, more preferably R⁵is 2-[(diethylamino)-2-hydroxyethyl]aminocarbonyl,2-(N-ethyl-N-2-hydroxyethylamino)ethylaminocarbonyl,carboxymethylamino-carbonyl, or2-(2-hydroxyethylamino)ethylamino-carbonyl.

(11) Yet another preferred group of compounds of Formula (I) is thatwherein R⁶ is —COR¹⁰ wherein R¹⁰ is —NR¹³R¹⁴ wherein R¹³ is hydrogen andR¹⁴ is lower alkyl substituted with hydroxy, lower alkyl substitutedwith hydroxyalkylamino, carboxy, or —NR¹⁸R¹⁹ wherein R¹⁸ and R¹⁹ areindependently hydrogen or lower unsubstituted alkyl; more preferably R⁶is [2-(diethylamino)-2-hydroxy]ethylaminocarbonyl,2-(N-ethyl-N-2-hydroxyethyl-amino)ethylaminocarbonyl,carboxymethylaminocarbonyl, or2-(2-hydroxyethylamino)ethylamino-carbonyl.

(12) Yet another preferred group of compounds of Formula (I) is thatwherein R⁵ is —COR¹⁰ wherein. R¹⁰ is —NR¹¹(CH₂)_(n)R¹² wherein R¹² is—N⁺(O⁻)NR¹³R¹⁴ or —N(OH)R¹³ wherein R¹³ and R¹⁴ are independentlyselected from the group consisting of hydrogen and unsubstituted loweralkyl, preferably R⁵ is 2-(N-hydroxy-N-ethylamino)-ethylaminocarbonyl or2-[N⁺(O⁻(C₂H₅)₂]ethyl-aminocarbonyl

(13) Yet another preferred group of compounds of Formula (I) is thatwherein R⁶ is —COR¹⁰ wherein R¹⁰ is —NR¹¹(CH₂)_(n)R¹² wherein R¹² is—N⁺(O⁻)NR¹³R¹⁴ or —N(OH)R¹³ wherein R¹³ and R¹⁴ are independentlyselected from the group consisting of hydrogen and unsubstituted loweralkyl, preferably R⁶ is 2-(N-hydroxy-N-ethylamino)ethylaminocarbonyl or2-[N⁺(O⁻)(C₂H₅)₂]ethyl-aminocarbonyl.

(14) In the above preferred groups (6)-(13) when R⁵ is —COR¹⁰, then amore preferred group of compounds is that wherein:

-   -   R⁶ is selected from the group consisting of hydrogen and alkyl,        preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl,        isobutyl, or n-butyl, more preferably hydrogen or methyl; and    -   R⁷ is selected from the group consisting of hydrogen, alkyl,        aryl, heteroaryl, and —C(O)R¹⁷ wherein R¹⁷ is hydroxy, alkyl or        aryl, more preferably hydrogen, methyl, ethyl, isopropyl, n-,        iso or tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more        preferably methyl, hydrogen or phenyl.

(15) In the above preferred groups; (6)-(13) when R⁵ is —COR¹⁰, thenanother more preferred group of compounds is that wherein R⁶ and R⁷combine to form —(CH₂)₄—.

(16) In the above preferred groups (6)-(13) when R⁶ is —COR¹⁰, then amore preferred group of compounds is that wherein:

-   -   R⁵ is selected from the group consisting of hydrogen and alkyl,        preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl,        isobutyl, or n-butyl, more preferably hydrogen or methyl; and    -   R⁷ is selected from the group consisting of hydrogen, alkyl,        aryl, heteroaryl, and —C(O)R¹⁷, wherein R¹⁷ is hydroxy, alkyl or        aryl, more preferably hydrogen, methyl, ethyl, isopropyl, n-,        iso or tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more        preferably methyl, hydrogen or phenyl.

(17) Within the above preferred and more preferred groups (6)-(16), aneven more preferred group of compounds is that wherein:

-   -   R¹ is hydrogen, alkyl, —C(O)NR⁸R⁹, unsubstituted cycloalkyl or        aryl, preferably hydrogen, phenyl,        3,4-dimethoxyphenylaminocarbonyl,        4-methoxy-3-chlorophenyl-aminocarbonyl, even more preferably        hydrogen or methyl, most preferably hydrogen;    -   R² is cyano, hydrogen, halo, lower alkoxy, aryl or —S(O)₂NR¹³R¹⁴        wherein R¹³ is hydrogen and R¹⁴ is hydrogen, aryl or alkyl,        preferably R² is hydrogen, chloro, bromo, fluoro, methoxy,        ethoxy, phenyl, dimethylaminosulfonyl,        3-chlorophenyl-aminosulfonyl, carboxy, methoxy, aminosulfonyl,        methylaminosulfonyl, phenylaminosulfonyl,        pyridin-3-yl-aminosulfonyl, dimethylaminosulfonyl,        isopropylamino-sulfonyl, more preferably hydrogen, fluoro, or        bromo;    -   R³ is selected from the group consisting of hydrogen, lower        alkoxy, —C(O)R¹⁵, —NR¹³C(O)R¹⁴, aryl preferably aryl optionally        substituted with one or two substitutents selected from the        group consisting of lower alkyl, halo, or lower alkoxy, and        heteroaryl, preferably heteroaryl optionally substituted with        one or two substitutents selected from the group consisting of        lower alkyl, halo, or lower alkoxy, preferably hydrogen,        methoxy, carboxy, phenyl, pyridin-3-yl, 3,4-dichlorophenyl,        2-methoxy-5-isopropylphenyl, 4-n-butylphenyl, 3-isopropylphenyl,        more preferably hydrogen or phenyl; and    -   R⁴ is hydrogen.

(18) Another more preferred group of compounds of Formula (I) is thatwherein:

-   -   R¹ is hydrogen, alkyl, —C(O)NR⁸R⁹, unsubstituted cycloalkyl or        aryl, preferably hydrogen, 3,4-dimethoxy-phenyl-aminocarbonyl,        4-methoxy-3-chlorophenylaminocarbonyl, even more preferably        hydrogen or methyl, particularly hydrogen;    -   R² cyano, hydrogen, halo, lower alkoxy, aryl or —S(O)₂NR¹³R¹⁴        wherein R¹³ is hydrogen and R¹⁴ is hydrogen, aryl or alkyl,        preferably R² is hydrogen, chloro, bromo, fluoro, methoxy,        ethoxy, phenyl, dimethylaminosulfonyl,        3-chlorophenyl-aminosulfonyl, carboxy, methoxy, aminosulfonyl,        methylaminosulfonyl, phenylaminosulfonyl,        pyridin-3-yl-aminosulfonyl, dimethylaminosulfonyl,        isopropylamino-sulfonyl, more preferably hydrogen, fluoro, or        bromo;    -   R³ is selected from the group consisting of hydrogen, lower        alkoxy, —C(O)R¹⁵, —NR¹³C(O)R¹⁴, aryl preferably aryl optionally        substituted with one or two substitutents selected from the        group consisting of lower alkyl, halo, or lower alkoxy, and        heteroaryl, preferably heteroaryl optionally substituted with        one or two substitutents selected from the group consisting of        lower alkyl, halo, or lower alkoxy,; preferably hydrogen,        methoxy, carboxy, phenyl, pyridin-3-yl, 3,4-dichlorophenyl,        2-methoxy-5-isopropyiphenyl, 4-n-butylphenyl, 3-isopropylphenyl,        more preferably hydrogen or phenyl; and    -   R⁴ is hydrogen.

Within the above preferred group (18) a more preferred group ofcompounds is wherein:

-   -   R⁵ is —COR¹⁰ where R¹⁰ is as defined in the Summary of the        Invention, preferably —NR¹¹(CH₂)_(n)R¹² or —NR¹³R¹⁴ as defined        in the Summary of the Invention.    -   R⁶ is selected from the group consisting of hydrogen and alkyl,        preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl,        isobutyl, or n-butyl, more preferably hydrogen or methyl; and    -   R⁷ is selected from the group consisting of hydrogen, alkyl,        aryl, heteroaryl, and —C(O)R¹⁷ wherein R¹⁷ is hydroxy, alkyl or        aryl, more preferably hydrogen, methyl, ethyl, isopropyl, n-,        iso or tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more        preferably methyl, hydrogen or phenyl.

In the above preferred group (18) another more preferred group ofcompounds is that wherein:

-   -   R⁶ is —COR¹⁰ where R¹⁰ is as defined in the Summary of the        Invention, preferably —NR¹¹(CH₂)_(n)R¹² or —NR¹³R¹⁴ as defined        in the Summary of the Invention.    -   R⁵ is selected from the group consisting of hydrogen and alkyl,        preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl,        isobutyl, or n-butyl, more preferably hydrogen or methyl; and    -   R⁷ is selected from the group consisting of hydrogen, alkyl,        aryl, heteroaryl, and —C(O)R¹⁷ wherein R¹⁷ is hydroxy, alkyl or        aryl, more preferably hydrogen, methyl, ethyl, isopropyl, n-,        iso or tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more        preferably methyl, hydrogen or phenyl.

(19) Another more preferred group of compounds of Formula (I) is thatwherein:

-   -   R¹ and R⁴ ate hydrogen;    -   R² is selected from the group consisting of hydrogen, halo,        lower alkoxy, —C(O)R¹⁵ and —S(O)₂NR¹³R¹⁴;    -   R³ is selected from the group consisting of hydrogen, lower        alkoxy, —C(O)R¹⁵, —S(O)₂NR¹³R¹⁴, aryl and heteroaryl;    -   R⁵ is —C(O)R¹⁰;    -   R⁶ is selected from the group consisting of hydrogen and lower        alkyl; and    -   R⁷ is selected from the group consisting of hydrogen, lower        alkyl and —C(O)R¹⁷.

It is another presently preferred embodiment of this invention that, ina compound having a structure as described in (15):

-   -   R¹⁰ is selected from the group consisting of hydroxy, lower        alkoxy and —NR¹¹(CH₂)_(n)R¹², wherein    -   n is 2 or 3;    -   R¹¹ is selected from the group consisting of hydrogen and lower        alkyl; and,    -   R¹² is selected from the group consisting of aryl and —NR¹³R¹⁴.

It is a further presently preferred embodiment of this invention that,in a compound having a structure as described in the previous twoparagraphs, R¹³ and R¹⁴ are independently selected from the groupconsisting of hydrogen, lower alkyl, and, combined, —(CH₂)₄—, —(CH₂)₅—,—CH₂)₂O(CH₂)₂— or —(CH₂)₂N(CH₃)(CH₂)₂—.

(20) Another presently preferred embodiment of this invention is acompound in which:

-   -   R¹ is selected from the group consisting of hydrogen, lower        alkyl, —(CH₂)_(r)R¹⁶ and —C(O)NR⁸R⁹;    -   R² is selected from the group consisting of hydrogen, halogen,        aryl and —S(O)₂NR¹³R¹⁴;    -   R³ is selected from the group consisting of hydrogen, lower        alkyl, lower alkoxy, aryl, heteroaryl and —C(O)R¹⁵;    -   R⁴ is hydrogen;    -   R⁵ is selected from the group consisting of hydrogen and lower        alkyl;    -   R⁶ is —C(O)R¹⁰;    -   R⁷ is selected from the group consisting of hydorgen, lower        alkyl and aryl;

R¹⁶ is selected from the group consisting of hydroxy and —C(O)R¹⁵; and,

-   -   r is 2 or 3.

A presently preferred embodiment of this invention is a compound havingas structure described in the paragraph just above in which R³ is aryloptionally substituted with one or more groups selected from the groupconsisting of lower alkyl, lower alkoxy and halo.

(21) Likewise, it is a presently preferred embodiment of this inventionthat, in a compound in which:

-   -   R¹ is selected from the group consisting of hydrogen, lower        alkyl, —(CH₂)_(r)R¹⁶ and —C(O)NR⁸R⁹;    -   R² is selected from the group consisting of hydrogen, halogen,        aryl and —S(O)₂NR¹³R¹⁴;    -   R³ is selected from the group consisting of hydrogen, lower        alkyl, lower alkoxy, aryl, heteroaryl and —C(O)R¹⁵;    -   R⁴ is hydrogen;    -   R⁵ is selected from the group consisting of hydrogen and lower        alkyl;    -   R⁶ is —C(O)R¹⁰;    -   R⁷ is selected from the group consisting of hydorgen, lower        alkyl and aryl;    -   R¹⁶ is selected from the group consisting of hydroxy and        —C(O)R¹⁵; and,    -   r is 2 or 3,    -   R¹⁰ is selected from the group consisting of hydroxy, lower        alkoxy, —NR¹³R¹⁴ and —NR¹¹(CH₂)_(n)R¹², wherein n is 1, 2 or 3,        R¹¹ is hydrogen and R¹² is selected from the group consisting of        hydroxy, lower alkoxy, —C(O)R¹⁵, heteroaryl and —NR¹³R¹⁴.

(22) A further presently preferred embodiment of this invention is acompound having a structure as described in the paragraph immediatelyabove in which R¹³ and R¹⁴ are independently selected from the groupconsisting of hydrogen, lower alkyl, heteroaryl and, combined, —(CH₂)₄—,—(CH₂)₅—, —(CH₂)₂O(CH₂)₂—, or —(CH₂)₂N(CH₃)(CH₂)₂—.

(23) Another presently preferred embodiment of this invention is acompound in which:

-   -   R¹ is —C(O)NR⁸R⁹, wherein R⁸ is hydrogen and R⁹ is aryl        optionally substituted with one or more groups selected from the        group consisting of halo, hydroxy and lower alkoxy;    -   R² is selected from the group consisting of hydrogen, halogen,        aryl and —S(O)₂NR¹³R¹⁴;    -   R³ is selected from the group consisting of hydrogen, lower        alkyl, lower alkoxy, aryl, heteroaryl and —C(O)R¹⁵;    -   R⁴ is hydrogen;    -   R⁵ is selected from the group consisting of hydrogen and lower        alkyl;    -   R⁶ is —C(O)R¹⁰;    -   R⁷ is selected from the group consisting of hydorgen, lower        alkyl and aryl;    -   R¹⁶ is selected from the group consisting of hydroxy and        —C(O)R¹⁵; and,    -   r is 2 or 3,

(24) A still further presently preferred embodiment of this invention isa compound in which:

-   -   R¹ is selected from the group consisting of hydrogen and lower        alkyl;    -   R² is selected from the group consisting of hydrogen, halo,        lower alkoxy, aryl, —C(O)R¹⁵ and —S(O)₂NR¹³R¹⁴;    -   R³ is selected from the group consisting of hydrogen, halo,        aryl, heteroaryl and —C(O)R¹⁵;    -   R⁴ is hydrogen;    -   R⁵ is —C(O)R¹⁰; and,    -   R⁶ and R⁷ combine to form a —(CH₂)₄— group.

In a compound having a structure as described in the paragraphimmediately above, it is a presently preferred embodiment that R¹⁰ isselected from the group consisting of hydroxy, alkoxy, —NR¹³R¹⁴ and—NH(CH₂)_(n)NR¹³R¹⁴ wherein n is 2 or 3.

It is a presently preferred embodiment of this invention that, in acompound having a structure as described in the two paragraphsimmediately above, R¹³ and R¹⁴ are independently selected from the groupconsisting of hydrogen, lower alkyl, and, combined, —(CH₂)₄—, —(CH₂)₅—,—(CH₂)₂O(CH₂)₂— or —(CH₂)₂N (CH₃)(CH₂)₂—.

Utility

The PKs whose catalytic activity is modulated by the compounds of thisinvention include protein tyrosine kinases of which there are two types,receptor tyrosine kinases (RTKs) and cellular tyrosine kinases (CTKs),and serine-threonine kinases (STKs). RTK mediated signal transduction isinitiated by extracellular interaction with a specific growth factor(ligand), followed by receptor dimerization, transient stimulation ofthe intrinsic protein tyrosine kinase activity and phosphorylation.Binding sites are thereby created for. intracellular signal transductionmolecules and lead to the formation of complexes with a spectrum ofcytoplasmic signaling molecules that facilitate the appropriate cellularresponse (e.g., cell division, metabolic effects on the extracellularmicroenvironment, etc.). See, Schlessinger and Ullrich, 1992, Neuron9:303-391.

It has been shown that tyrosine phosphorylation sites on growth factorreceptors function as high-affinity binding sites for SH2 (src homology)domains of signaling molecules. Fantl et al., 1992, Cell 69:413-423,Songyang et al., 1994, Mol. Cell. Biol. 14:2777-2785), Songyang et al.,1993, Cell 72:767-778, and Koch et al., 1991, Science 252:668-678.Several intracellular substrate proteins that associate with RTKs havebeen identified. They may be divided into two principal groups: (1)substrates that have a catalytic domain, and (2) substrates which lacksuch domain but which serve as adapters and associate with catalyticallyactive molecules. Songyang et al., 1993, Cell 72:767-778. Thespecificity of the interactions between receptors and SH2 domains oftheir substrates is determined by the amino acid residues immediatelysurrounding the phosphorylated tyrosine residue. Differences in thebinding affinities between SH2 domains and the amino acid sequencessurrounding the phosphotyrosine residues on particular receptors areconsistent with the observed differences in their substratephosphorylation profiles. Songyang et al., 1993, Cell 72:767-778. Theseobservations suggest that the function of each RTK is determined notonly by its pattern of expression and ligand availability but also bythe array of downstream signal transduction pathways that are activatedby a particular receptor. Thus, phosphorylation provides an importantregulatory step which determines the selectivity of signaling pathwaysrecruited by specific growth factor receptors, as well asdifferentiation factor receptors.

STKs, being primarily cytosolic, affect the internal biochemistry of thecell, often as a down-line response to a PTK event. STKs have beenimplicated in the signaling process which initiates DNA synthesis andsubsequent mitosis leading to cell proliferation.

Thus, PK signal transduction results in, among other responses, cellproliferation, differentiation, growth and metabolism. Abnormal cellproliferation may result in a wide array of disorders and diseases,including the development of neoplasia such as carcinoma, sarcoma,glioblastoma and hemangioma, disorders such as leukemia, psoriasis,arteriosclerosis, arthritis and diabetic retinopathy and other disordersrelated to uncontrolled angiogenesis and/or vasculogenesis.

A precise understanding of the mechanism by which the compounds of thisinvention inhibit PKs is not required in order to practice the presentinvention. However, while not hereby being bound to any particularmechanism or theory, it is believed that the compounds interact with theamino acids in the catalytic region of PKs. PKs typically possess abi-lobate structure wherein ATP appears to bind in the cleft between thetwo lobes in a region where the amino acids are conserved among PKs.Inhibitors of PKs are believed to bind by non-covalent interactions suchas hydrogen bonding, van der Waals forces and ionic interactions in thesame general region where the aforesaid ATP binds to the PKs. Morespecifically, it is thought that the 2-indolinone component of thecompounds of this invention binds in the general space normally occupiedby the adenine ring of ATP. Specificity of a particular molecule for aparticular PK may then arise as the result of additional interactionsbetween the various substituents on the 2-indolinone core and the aminoacid domains specific to particular PKs. Thus, different indolinonesubstituents may contribute to preferential binding to particular PKs.The ability to select compounds active at different ATP (or othernucleotide) binding sites makes the compounds of this invention usefulfor targeting any protein with such a site. The compounds disclosedherein thus have utility in in vitro assays for such proteins as well asexhibiting in vivo therapeutic effects through interaction with suchproteins.

Additionally, the compounds of the present invention provide atherapeutic approach to the treatment of many kinds of solid tumors,including but not limited to carcinomas, sarcomas including Kaposi'ssarcoma, erythroblastoma, glioblastoma, meningioma, astrocytoma,melanoma and myoblastoma. Treatment or prevention of non-solid tumorcancers such as leukemia are also contemplated by this invention.Indications may include, but are not limited to brain cancers, bladdercancers, ovarian cancers, gastric cancers, pancreas cancers, coloncancers, blood cancers, lung cancers and bone cancers.

Further examples, without limitation, of the types of disorders relatedto inappropriate PK activity that the compounds described herein may beuseful in preventing, treating and studying, are cell proliferativedisorders, fibrotic disorders and metabolic disorders.

Cell proliferative disorders, which may be prevented, treated or furtherstudied by the present invention include cancer, blood vesselproliferative disorders and mesangial cell proliferative disorders.

Blood vessel proliferative disorders refer to disorders related toabnormal vasculogenesis (blood vessel formation) and angiogenesis(spreading of blood vessels). While vasculogenesis and angiogenesis playimportant roles in a variety of normal physiological processes such asembryonic development, corpus luteum formation, wound healing and organregeneration, they also play a pivotal role in cancer development wherethey result in the formation of new capillaries needed to keep a tumoralive. Other examples of blood vessel proliferation disorders includearthritis, where new capillary blood vessels invade the joint anddestroy cartilage, and ocular diseases, like diabetic retinopathy, wherenew capillaries in the retina invade the vitreous, bleed and causeblindness.

Two structurally related RTKs have been identified to bind VEGF withhigh affinity: the fms-like tyrosine 1 (fit-1) receptor (Shibuya et al.,1990, Oncogene,5:519-524; De Vries et al., 1992, Science, 255:989-991)and the KDR/FLK-1 receptor, also known as VEGF-R2. Vascular endothelialgrowth factor (VEGF) has been reported to be an endothelial cellspecific mitogen with in vitro endothelial cell growth promotingactivity. Ferrara & Henzel, 1989, Biochein. Biophys. Res. Comm.,161:851-858; Vaisman et al., 1990, J. Biol. Chem., 265:19461-19566.Information set forth in U.S. application Ser. Nos. 08/193,829,08/038,596 and 07/975,750, strongly suggest that VEGF is not onlyresponsible for endothelial cell proliferation, but also is the primeregulator of normal and pathological angiogenesis. See generally,Klagsburn & Soker, 1993, Current Biology, 3(10)699-702; Houck, et al.,1992, J. Biol. Chem., 267:26031-26037.

Normal vasculogenesis and angiogenesis play important roles in a varietyof physiological processes such as embryonic development, wound healing,organ regeneration and female reproductive processes such as follicledevelopment in the corpus luteum during ovulation and placental growthafter pregnancy. Folkman & Shing, 1992, J. Biological Chem.,267(16):10931-34. Uncontrolled vasculogenesis and/or angiogenesis hasbeen associated with diseases such as, diabetes as well as withmalignant solid tumors that rely on vascularization for growth.Klagsburn & Soker, 1993, Current Biology, 3(10):699-702; Folkham, 1991,J. Natl. Cancer Inst., 82:4-6; Weidner, et al., 19.91, New Engl. J.Med., 324:1-5.

The surmised role of VEGF in endothelial cell proliferation andmigration during angiogenesis and vasculogenesis indicates an importantrole for the KDR/FLK-1 receptor in these processes. Diseases such asdiabetes mellitus (Folkman, 198, in XIth Congress of Thrombosis andHaemostasis (Verstraeta, et al., eds.), pp. 583-596, Leuven UniversityPress, Leuven) and arthritis, as well as malignant tumor growth mayresult from uncontrolled angiogenesis. See e.g., Folkman, 1971, N. Engl.J. Med., 285:1182-1186. The receptors to which VEGF specifically bindsare an important and powerful therapeutic target for the regulation andmodulation of vasculogenesis and/or angiogenesis and a variety of severediseases which involve abnormal cellular growth caused by suchprocesses. Plowman, et al., 1994, DN&P, 7 (6):334-339. Moreparticularly, the KDR/FLK-1 receptor's highly specific role inneovascularization make it a choice target for therapeutic approaches tothe treatment of cancer and other diseases which involve theuncontrolled formation of blood vessels.

Thus, the present invention provides compounds capable of regulatingand/or modulating tyrosine kinase signal transduction includingKDR/FLK-1 receptor signal transduction in order to inhibit or promoteangiogenesis and/or vasculogenesis, that is, compounds that inhibit,prevent, or interfere with the signal transduced by KDR/FLK-1 whenactivated by ligands such as VEGF. Although it is believed that thecompounds of the present invention act on a receptor or other componentalong the tyrosine kinase signal transduction pathway, they may also actdirectly on the tumor cells that result from uncontrolled angiogenesis.

Although the nomenclature of the human and murine counterparts of thegeneric “flk-I” receptor differ, they are, in many respects,interchangeable. The murine receptor, Flk-1, and its human counterpart,KDR, share a sequence homology of 93.4% within the intracellular domain.Likewise, murine FLK-I binds human VEGF with the same affinity as mouseVEGF, and accordingly, is activated by the ligand derived from eitherspecies. Millauer et al., 1993, Cell, 72:835-846; Quinn et al., 1993,Proc. Natl. Acad. Sci. USA, 90:7533-7537. FLK-1 also associates with andsubsequently tyrosine phosphorylates human RTK substrates (e.g., PLC-γor p85) when co-expressed in 293 cells (human embryonal kidneyfibroblasts).

Models which rely upon the FLK-1 receptor therefore are directlyapplicable to understanding the KDR receptor. For example, use of themurine FLK-1 receptor in methods which identify compounds that regulatethe murine signal transduction pathway are directly applicable to theidentification of compounds which may be used to regulate the humansignal transduction pathway, that is, which regulate activity related tothe KDR receptor. Thus, chemical compounds identified as inhibitors ofKDR/FLK-1 in vitro, can be confirmed in suitable in vivo models. Both invivo mouse and rat animal models have been demonstrated to be ofexcellent value for the examination of the clinical potential of agentsacting on the KDR/FLK-1 induced signal transduction pathway.

Thus, the present invention provides compounds that regulate, modulateand/or inhibit vasculogenesis and/or angiogenesis by affecting theenzymatic activity of the KDR/FLK-1 receptor and interfering with thesignal transduced by KDR/FLK-1. Thus the present invention provides atherapeutic approach to the treatment of many kinds of solid tumorsincluding, but not limited to, glioblastoma, melanoma and Kaposi'ssarcoma, and ovarian, lung, mammary, prostate, pancreatic, colon andepidermoid carcinoma. In addition, data suggests the administration ofcompounds which inhibit the KDR/Flk-1 mediated signal transductionpathway may also be used in the treatment of hemangioma, restenois anddiabetic retinopathy.

Furthermore, this invention relates to the inhibition of vasculogenesisand angiogenesis by other receptor-mediated pathways, including thepathway comprising the flt-1 receptor.

Receptor tyrosine kinase mediated signal transduction is initiated byextracellular interaction with a specific growth factor (ligand),followed by receptor dimerization, transient stimulation of theintrinsic protein tyrosine kinase activity and autophosphorylation.Binding sites are thereby created for intracellular signal transductionmolecules which leads to the formation of complexes with a spectrum ofcytoplasmic signalling molecules that facilitate the appropriatecellular response, e.g., cell division and metabolic effects to theextracellular microenvironment. See, Schlessinger and Ullrich, 1992,Neuron, 9:1-20.

The close homology of the intracellular regions of KDR/FLK-1 with thatof the PDGF-β receptor (50.3% homology) and/or the related flt-1receptor indicates the induction of overlapping signal transductionpathways. For example, for the PDGF-β receptor, members of the srcfamily (Twamley et al., 1993, Proc. Natl. Acad. Sci. USA, 90:7696-7700),phosphatidylinositol-3′-kinase (Hu et al., 1992, Mol. Cell. Biol.,12:981-990), phospholipase cγ (Kashishian & Cooper, 1993, Mol. Cell.Biol., 4:49-51), ras-GTPase-activating protein, (Kashishian et al.,1992, EMBO J., 11:1373-1382), PTP-ID/syp (Kazlauskas et al., 1993, Proc.Natl. Acad. Sci. USA, 10 90:6939-6943), Grb2 (Arvidsson et al., 1994,Mol. Cell. Biol., 14:6715-6726), and the adapter molecules Shc and Nck(Nishimura et al., 1993, Mol. Cell. Biol., 13:6889-6896), have beenshown to bind to regions involving different autophosphorylation sites.See generally, Claesson-Welsh, 1994, Prog. Growth Factor Res., 5:37-54.Thus, it is likely that signal transduction pathways activated byKDR/FLK-1 include the ras pathway (Rozakis et al., 1992, Nature,360:689-692), the PI-3′-kinase, the src-mediated and the plcγ-mediatedpathways. Each of these pathways may play a critical role in theangiogenic and/or vasculogenic effect of KDR/FLK-1 in endothelial cells.Consequently, a still further aspect of this invention relates to theuse of the organic compounds described herein to modulate angiogenesisand vasculogenesis as such processes are controlled by these pathways.

Conversely, disorders related to the shrinkage, contraction or closingof blood vessels, such as restenosis, are also implicated and may betreated or prevented by the methods of this invention.

Fibrotic disorders refer to the abnormal formation of extracellularmatrices. Examples of fibrotic disorders include hepatic cirrhosis andmesangial cell proliferative disorders. Hepatic cirrhosis ischaracterized by the increase in extracellular matrix constituentsresulting in the formation of a hepatic scar. An increased extracellularmatrix resulting in a hepatic scar can also be caused by a viralinfection such as hepatitis. Lipocytes appear to play a major role inhepatic cirrhosis. Other fibrotic disorders implicated includeatherosclerosis.

Mesangial cell proliferative disorders refer to disorders brought aboutby abnormal proliferation of mesangial cells. Mesangial proliferativedisorders include various human renal diseases such asglomerulonephritis, diabetic nephropathy and malignant nephrosclerosisas well as such disorders as thrombotic microangiopathy syndromes,transplant rejection, and glomerulopathies. The RTK PDGFR has beenimplicated in the maintenance of mesangial cell proliferation. Floege etal., 1993, Kidney International 43:47S-54S.

Many cancers are cell proliferative disorders and, as noted previously,PKs have been associated with cell proliferative disorders. Thus, it isnot surprising that PKs such as, for example, members of the RTK-familyhave been associated with the development of cancer. Some of thesereceptors, like EGFR (Tuzi et al., 1991, Br. J. Cancer 63:227-233, Torpet al., 1992, APMIS 100:713-719) HER2/neu (Slamon et al., 1989, Science244:707-712) and PDGF-R (Kumabe et al., 1992, Oncogene, 7:627-633) areover-expressed in many tumors and/or persistently activated by autocrineloops. In fact, in the most common and severe cancers these receptorover-expressions (Akbasak and Suner-Akbasak et al., 1992, J. Neurol.Sci., 111:119-133, Dickson et al., 1992, Cancer Treatment Res.61:249-273, Korc et al., 1992, J. Clin. Invest. 90:1352-1360) andautocrine loops (Lee and Donoghue, 1992, J. Cell. Biol., 118:1057-1070,Korc et al., supra, Akbasak and Suner-Akbasak et al., supra) have beendemonstrated. For example, EGFR has been associated with squamous cellcarcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancerand bladder cancer. HER2 has been associated with breast, ovarian,gastric, lung, pancreas and bladder cancer. PDGFR has been associatedwith glioblastoma andmelanoma as well as lung, ovarian and prostatecancer. The RTK c-met has also been associated with malignant tumorformation. For example, c-met has been associated with, among othercancers, colorectal, thyroid, pancreatic, gastric and hepatocellularcarcinomas and lymphomas. Additionally c-met has been linked toleukemia. Over-expression of the c-met gene has also been detected inpatients with Hodgkins disease and Burkitts disease.

IGF-IR, in addition to being implicated in nutritional support and intype-II diabetes, has also been associated with several types ofcancers. For example, IGF-I has been implicated as an autocrine growthstimulator for several tumor types, e.g. human breast cancer carcinomacells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and smalllung tumor cells (Macauley et al., 1990, Cancer Res., 50:2511-2517). Inaddition, IGF-I, while integrally involved in the normal growth anddifferentiation of the nervous system, also appears to be an autocrinestimulator of human gliomas. Sandberg-Nordqvist et al., 1993, CancerRes. 53:2475-2478. The importance of IGF-IR and its ligands in cellproliferation is further supported by the fact that many cell types inculture (fibroblasts, epithelial cells, smooth muscle cells,T-lymphocytes, myeloid cells, chondrocytes and osteoblasts (the stemcells of the bone marrow)) are stimulated to grow by IGF-I. Goldring andGoldring, 1991, Eukaryotic Gene Expression,1:301-326. Baserga andCoppola suggest that IGF-IR plays a central role in the mechanism oftransformation and, as such, could be a preferred target for therapeuticinterventions for a broad spectrum of human malignancies. Baserga, 1995,Cancer Res., 55:249-252, Baserga, 1994, Cell 79:927-930, Coppola et al.,1994, Mol. Cell. Biol., 14:4588-4595.

STKs have been implicated in many types of cancer including, notably,breast cancer (Cance, et al., Int. J. Cancer, 54:571-77 (1993)).

The association between abnormal PK activity and disease is notrestricted to cancer. For example, RTKs have been associated withdiseases such as psoriasis, diabetes mellitus, endometriosis,angiogenesis, atheromatous plaque development, Alzheimer's disease,restenosis, von Hippel-Lindau disease, epidermal hyperproliferation,neurodegenerative diseases, age-related macular degeneration andhemangiomas. For example, EGFR has been indicated in corneal and dermalwound healing. Defects in Insulin-R and IGF-1R are indicated in type-IIdiabetes mellitus. A more complete correlation between specific RTKs andtheir therapeutic indications is set forth in Plowman et al., 1994,DN&P. 7:334-339.

As noted previously, not only RTKs but CTKs including, but, not limitedto, src, abl, fps, yes, fyn, lyn lck, blk, hck, fgr and yrk (reviewed byBolen et al., 1992, FASEB J., 6:3403-3409) are involved in theproliferative and metabolic signal transduction pathway and thus couldbe expected, and have been shown, to be involved in many PTK-mediateddisorders to which the present invention is directed. For example,mutated src (v-src) has been shown to be an oncoprotein (pp60^(v-src))in chicken. Moreover, its cellular homolog, the proto-oncogenepp60^(c-src) transmits oncogenic signals of many receptors.Over-expression of EGFR or HER2/neu in tumors leads to the constitutiveactivation of pp60^(c□src), which is characteristic of malignant cellsbut absent in normal cells. On the other hand, mice deficient in theexpression of c-src exhibit an osteopetrotic phenotype, indicating a keyparticipation of c-src in osteoclast function and a possible involvementin related disorders.

Similarly, Zap70 has been implicated in T-cell signaling which mayrelate to autoimmune disorders.

STKs have been associated with inflamation, autoimmune disease,immunoresponses, and hyperproliferation disorders such as restenosis,fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis.

PKs have also been implicated in embryo implantation. Thus, thecompounds of this invention may provide an effective method ofpreventing such embryo implantation and thereby be useful as birthcontrol agents. Additional disorders which may be treated or preventedusing the compounds of this invention are immunological disorders suchas autoimmune disease, AIDS and cardiovasular disorders such asatherosclerosis.

Finally, both RTKs and CTKs are currently suspected as being involved inhyperimmune disorders.

Examples of the effect of a number of exemplary compounds of thisinvention on several PTKs are shown in Table 2 below. The compounds anddata presented are not to be construed as limiting the scope of thisinvention in any manner whatsoever.

Administration and Pharmaceutical Composition

A compound of the present invention or a phearmaceutically acceptablesalt thereof, can be administered as such to a human patient or can beadministered in pharmaceutical compositions in which the foregoingmaterials are mixed with suitable carriers or excipient(s). Techniquesfor formulation and administration of drugs may be found in “Remington'sPharmacological Sciences,” Mack Publishing Co., Easton, Pa., latestedition.

As used herein, “administer” or “administration” refers to the deliveryof a compound of Formula (I) or a pharmaceutically acceptable saltthereof or of a pharmaceutical composition containing a compound ofFormula (I) or a pharmaceutically acceptable salt thereof of thisinvention to an organism for the purpose of prevention or treatment of aPK-related disorder.

Suitable routes of administration may include, without limitation, oral,rectal, transmucosal or intestinal administration or intramuscular,subcutaneous, intramedullary, intrathecal, direct intraventricular,intravenous, intravitreal, intraperitoneal, intranasal, or intraocularinjections. The preferred routes of administration are oral andparenteral.

Alternatively, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a solid tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tumor-specific antibody.The liposomes will be targeted to and taken up selectively by the tumor.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated by combiningthe active compounds with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the compounds of the invention tobe formulated as tablets, pills, lozenges, dragees, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient. Pharmaceutical preparations for oral use can be made using asolid excipient, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding other suitableauxiliaries if desired, to obtain tablets or dragee cores. Usefulexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol, cellulose preparations such as,for example, maize starch, wheat starch, rice starch and potato starchand other materials such as gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinyl-pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginicacid. A salt such as sodium alginate may also be used.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with a fillersuch as lactose, a binder such as starch, and/or a lubricant such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. Stabilizers may be added in these formulations, also.

Pharmaceutical compositions which may also be used include hard gelatincapsules. As a non-limiting example, the active compound capsule oraldrug product formulation may be as 50 and 200 mg dose strengths(formulation codes J-011248-AA-00 and J-011248-AA-01, respectively). Thetwo dose strengths are made from the same granules by filling intodifferent size hard gelatin capsules, size 3 for the 50 mg capsule andsize 0 for the 200 mg capsule. The composition of the formulation maybe, for example, as indicated in Table 2. TABLE 2 Concentration Amountin Amount in Ingredient in Granulation 50 mg 200 mg Name/Grade (% w/w)Capsule (mg) Capsule (mg) Formulation J-011248-AA J-011248-AA-00J-011248-AA-01 Code Active 65.0 50.0 200.0 Compound NF Mannitol NF 23.518.1 72.4 Croscarmellose 6.0 4.6 18.4 sodium NF Povidone 5.0 3.8 15.2 K30 NF Magnesium 0.5 0.38 1.52 stearate NF Capsule, Size 3 Size 0 Swedishyellow NF

The capsules may be packaged into brown glass or plastic bottles toprotect the active compound from light. The containers containing theactive compound capsule formulation must be stored at controlled roomtemperature (15-30° C.).

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray using a pressurized pack or a nebulizer and a suitable propellant,e.g., without limitation, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetra-fluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be controlled byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The compounds may also be formulated for parenteral administration,e.g., by bolus injection or continuous infusion. Formulations forinjection may be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulating materials such assuspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of a water soluble form, such as, without limitation,a salt, of the active compound. Additionally, suspensions of the activecompounds may be prepared in a lipophilic vehicle. Suitable lipophilicvehicles include fatty oils such as sesame oil, synthetic fatty acidesters such as ethyl oleate and triglycerides, or materials suchasliposomes. Aqueous injection suspensions may contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers and/or agents that increase the solubilityof the compounds to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, using, e.g., conventional suppositorybases such as cocoa butter or other glycerides.

In addition to the fomulations described previously, the compounds mayalso be formulated as depot preparations. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. A compound of thisinvention may be formulated for this route of administration withsuitable polymeric or hydrophobic materials (for instance, in anemulsion with a pharamcologically acceptable oil), with ion exchangeresins, or as a sparingly soluble derivative such as, withoutlimitation, a sparingly soluble salt.

A non-limiting example of a pharmaceutical carrier for the hydrophobiccompounds of the invention is a cosolvent system comprising benzylalcohol, a nonpolar surfactant, a water-miscible organic polymer and anaqueous phase such as the VPD co-solvent system. VPD is a solution of 3%w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80,and 65% w/v polyethylene glycol 300, made up to volume in absoluteethanol. The VPD co-solvent system (VPD:D5W) consists of VPD diluted 1:1with a 5% dextrose in water solution. This co-solvent system dissolveshydrophobic compounds well, and itself produces low toxicity uponsystemic administration. Naturally, the proportions of such a co-solventsystem may be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the co-solventcomponents may be varied: for example, other low-toxicitynonpolarsurfactants may be used instead of Polysorbate 80, the fraction size ofpolyethylene glycol may be varied, other biocompatible polymers mayreplace polyethylene glycol, e.g., polyvinyl pyrrolidone, and othersugars or polysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. Inaddtion, certain organic solvents such as dimethylsulfoxide also may beemployed, although often at the cost of greater toxicity.

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained-release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

The pharmaceutical compositions herein also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include, but are not limited to, calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

Many of the PK modulating compounds of the invention may be provided asphysiologically acceptable salts wherein the claimed compound may formthe negatively or the positively charged species. Examples of salts inwhich the compound forms the positively charged moiety include, withoutlimitation, quaternary ammonium (defined elsewhere herein), salts suchas the hydrochloride, sulfate, carbonate, lactate, tartrate, malate,maleate, succinate wherein the nitrogen atom of the quaternary ammoniumgroup is a nitrogen of the selected compound of this invention which hasreacted with the appropriate acid. Salts in which a compound of thisinvention forms the negatively charged species include, withoutlimitation, the sodium, potassium, calcium and magnesium salts formed bythe reaction of a carboxylic acid group in the compound with anappropriate base (e.g. sodium hydroxide (NaOH), potassium hydroxide(KOH), Calcium hydroxide (Ca(OH)₂), etc.).

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in anamount sufficient to achieve the intended purpose, e.g., the modulationof PK activity or the treatment or prevention of a PK-related disorder.

More specifically, a therapeutically effective amount means an amount ofcompound effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compound used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromcell culture assays. Then, the dosage can be formulated for use inanimal models so as to achieve a circulating concentration range thatincludes the IC₅₀ as determined in cell culture (i.e., the concentrationof the test compound which achieves a half-maximal inhibition of the PKactivity). Such information can then be used to more accuratelydetermine useful doses in humans.

Toxicity and therapeutic efficacy of the compounds described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the IC₅₀ and the LD₅₀ (bothof which are discussed elsewhere herein) for a subject compound. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage mayvary depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p.1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active species which are sufficient to maintain thekinase modulating effects. These plasma levels are referred to asminimal effective concentrations (MECs). The MEC will vary for eachcompound but can be estimated from in vitro data, e.g., theconcentration necessary to achieve 50-90% inhibition of a kinase may beascertained using the assays described herein. Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration. HPLC assays or bioassays can be used to determine plasmaconcentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen that maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

At present, the therapeutically effective amounts of compounds ofFormula (I) may range from approximately 25 mg/m² to 1500 mg/m² per day;preferably about 3 mg/m²/day. Even more preferably 50mg/qm qd till 400mg/qd.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration and other procedures known in the art may be employed todetermine the correct dosage amount and interval.

The amount of a composition administered will, of course, be dependenton the subject being treated, the severity of the affliction, the mannerof administration, the judgment of the prescribing physician, etc.

The compositions may, if desired, be presented in a pack or dispenserdevice, such as an FDA approved kit, which may contain one or more unitdosage forms containing the active ingredient. The pack may for examplecomprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.The pack or dispenser may also be accompanied by a notice associatedwith the container in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals, which noticeis reflective of approval by the agency of the form of the compositionsor of human or veterinary administration. Such notice, for example, maybe of the labeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a compound of the invention formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition. Suitableconditions indicated on the label may include treatment of a tumor,inhibition of angiogenesis, treatment of fibrosis, diabetes, and thelike.

It is also an aspect of this invention that a compound described herein,or its salt or prodrug, might be combined with other chemotherapeuticagents for the treatment of the diseases and disorders discussed above.For instance, a compound, salt or prodrug of this invention might becombined with alkylating agents such as fluorouracil (5-FU) alone or infurther combination with leukovorin; or other alkylating agents such as,without limitation, other pyrimidine analogs such as UFT, capecitabine,gemcitabine and cytarabine, the alkyl sulfonates, e.g., busulfan (usedin the treatment of chronic granulocytic leukemia), improsulfan andpiposulfan; aziridines, e.g., benzodepa, carboquone, meturedepa anduredepa; ethyleneimines and methylmelamines, e.g., altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylolmelamine; and the nitrogenmustards, e.g., chlorambucil (used in the treatment of chroniclymphocytic leukemia, primary macroglobulinemia and non-Hodgkin'slymphoma), cyclophosphamide (used in the treatment of Hodgkin's disease,multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lungcancer, Wilm's tumor and rhabdomyosarcoma); estramustine, ifosfamide,novembrichin, prednimustine and uracil mustard (used in the treatment ofprimary thrombocytosis, non-Hodgkin's lymphoma, Hodgkin's disease andovarian cancer); and triazines, e.g., dacarbazine (used in the treatmentof soft tissue sarcoma).

A compound, salt or prodrug of this invention can also be used incombination with other antimetabolite chemotherapeutic agents such as,without limitation, folic acid analogs, e.g. methotrexate (used in thetreatment of acute lymphocytic leukemia, choriocarcinoma, mycosisfungiodes breast cancer, head and neck cancer and osteogenic sarcoma)and pteropterin; and the purine analogs such as mercaptopurine andthioguanine which find use in the treatment of acute granulocytic, acutelymphocytic and chronic granulocytic leukemias.

It is contemplated that a compound, salt or prodrug of this inventioncan also be used in combination with natural product basedchemotherapeutic agents such as, without limitation, the vincaalkaloids, e.g., vinblastin (used in the treatment of breast andtesticular cancer), vincristine and vindesine; the epipodophylotoxins,e.g., etoposide and teniposide, both of which are useful in thetreatment of testicular cancer and Kaposi's sarcoma; the antibioticchemotherapeutic agents, e.g., daunorubicin, doxorubicin, epirubicin,mitomycin (used to treat stomach, cervix, colon, breast, bladder andpancreatic cancer), dactinomycin, temozolomide, plicamycin, bleomycin(used in the treatment of skin, esophagus and genitourinary tractcancer); and the enzymatic chemotherapeutic agents such asL-asparaginase.

In addition to the above, a compound, salt or prodrug of this inventioncould also be used in combination with the platinum coordinationcomplexes (cisplatin, etc.); substituted ureas such as hydroxyurea;methylhydrazine derivatives, e.g., procarbazine; adrenocorticalsuppressants, e.g., mitotane, aminoglutethimide; and hormone and hormoneantagonists such as the adrenocorticosteriods (e.g., prednisone),progestins (e.g., hydroxyprogesterone caproate); estrogens (e.g.,diethylstilbesterol); antiestrogens such as tamoxifen; androgens, e.g.,testosterone propionate; and aromatase inhibitors such as anastrozole.

Finally, it is also contemplated that the combination of a compound ofthis invention will be effective in combination with mitoxantrone orpaclitaxel for the treatment of solid tumor cancers or leukemias suchas, without limitation, acute myelogenous (non-lymphocytic) leukemia.

General Synthetic Procedure

The following general methodology maybe employed to prepare thecompounds of this invention:

The appropriately substituted 2-oxindole (1 equiv.), the appropriatelysubstituted aldehyde (1.2 equiv.) and a base (0.1 equiv.) are mixed in asolvent (1-2 ml/mmol 2-oxindole) and the mixture is then heated for fromabout 2 to about 12 hours. After cooling, the precipitate that forms isfiltered, washed with cold ethanol or ether and vacuum dried to give thesolid product. If no precipitate forms, the reaction mixture isconcentrated and the residue is triturated with dichloromethane/ether,the resulting solid is collected by filtration and then dried. Theproduct may optionally be further purified by chromatography.

The base may be an organic or an inorganic base. If an organic base isused, preferably it is a nitrogen base. Examples of organic nitrogenbases include, but are not limited to, diisopropylamine, trimethylamine,triethylamine, aniline, pyridine, 1,8-diazabicyclo[5.4.1]undec-7-ene,pyrrolidine and piperidine.

Examples of inorganic bases are, without limitation, ammonia, alkalimetal or alkaline earth hydroxides, phosphates, carbonates,bicarbonates, bisulfates and amides. The alkali metals include, lithium,sodium and potassium while the alkaline earths include calcium,magnesium and barium.

In a presently preferred embodiment of this invention, when the solventis a protic solvent, such as water or alcohol, the base is an alkalimetal or an alkaline earth inorganic base, preferably, a alkali metal oran alkaline earth hydroxide.

It will be clear to those skilled in the art, based both on knowngeneral principles of organic synthesis and on the disclosures hereinwhich base would be most appropriate for the reaction contemplated.

The solvent in which the reaction is carried out may be a protic or anaprotic solvent, preferably it is a protic solvent. A “protic solvent”is a solvent which has hydrogen atom(s) covalently bonded to oxygen ornitrogen atoms which renders the hydrogen atoms appreciably acidic andthus capable of being “shared” with a solute through hydrogen bonding.Examples of protic solvents include, without limitation, water andalcohols.

An “aprotic solvent” may be polar or non-polar but, in either case, doesnot contain acidic hydrogens and therefore is not capable of hydrogenbonding with solutes. Examples, without limitation, of non-polar aproticsolvents, are pentane, hexane, benzene, toluene, methylene chloride andcarbon tetrachloride. Examples of polar aprotic solvents are chloroform,tetrahydrofuran, dimethylsulfoxide and dimethylformamide.

In a presently preferred embodiment of this invention, the solvent is aprotic solvent, preferably water or an alcohol such as ethanol.

The reaction is carried out at temperatures greater than roomtemperature. The temperature is generally from about 30° C. to about150° C., preferably about 80° C. to about 100° C., most preferable about75° C. to about 85° C., which is about the boiling point of ethanol. By“about” is meant that the temperature range is preferably within 10degrees Celcius of the indicated temperature, more preferably within 5degrees Celcius of the indicated temperature and, most preferably,within 2 degrees Celcius of the indicated temperature. Thus, forexample, by “about 75° C.” is meant 75° C.±10° C., preferably 75° C.±5°C. and most preferably, 75° C.±2° C.

2-Oxindoles and aldehydes, may be readily synthesized using techniqueswell known in the chemical arts. It will be appreciated by those skilledin the art that other synthetic pathways for forming the compounds ofthe invention are available and that the following is offered by way ofexample and not limitation.

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

Synthetic Examples

Method A: Formylation of Pyrroles

POCl₃ (1.1 equiv.) is added dropwise to dimethylformamide (3 equiv.) at−10° C. followed by addition of the appropriate pyrrole dissolved indimethylformamide. After stirring for two hours, the reaction mixture isdiluted with H₂O and basified to pH 11 with 10 N KOH. The precipitatewhich forms is collected by filtration, washed with H₂O and dried in avacuum oven to give the desired aldehyde.

Method B: Saponification of Pyrrolecarboxylic Acid Esters

A mixture of a pyrrolecarboxylic acid ester and KOH (2-4 equiv.) in EtOHis refluxed until reaction completion is indicated by thin layerchromatography (TLC). The cooled reaction mixtrue is acidified to pH 3with 1 N HCl. The precipitate which forms is collected by filtration,washed with H₂O and dried in a vacuum oven to give the desiredpyrrolecarboxylic acid.

Method C: Amidation

To a stirred solution of a pyrrolecarboxylic acid dissolved indimethylformamide(0.3M) is added1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (1.2 equiv.),1-hydroxybenzotriazole (1.2 equiv.), and triethylamine (2 equiv.). Theappropriate amine is added (1 equiv.) and the reaction stirred untilcompletion is indicated by TLC. Ethyl ;acetate is then added to thereaction mixture and the solution washed with saturated NaHCO₃ and brine(with extra salt), dried over anhydrous MgSO₄ and concentrated to affordthe desired amide.

Method D: Condensation of Aldehydes and Oxindoles Containing CarboxylicAcid Substituents

A mixture of the oxindole (1 equivalent), 1 equivalent of the aldehydeand 1-3 equivalents of piperidine (or pyrrolidine) in ethanol (0.4 M) isstirred at 90-100° C. until reaction completion is indicated by TLC. Themixture is then concentrated and the residue acidified with 2N HCl. Theprecipitate that forms is washed with H₂O and EtOH and then dried in avacuum oven to give the product.

Method E: Condensation of Aldehydes and Oxindoles not ContainingCarboxylic Acid Substituents

A mixture of the oxindole (1 equivalent), 1 equivalent of the aldehydeand 1-3 equivalents of piperidine (or pyrrolidine) in ethanol (0.4 M) isstirred at 90-100° C. until reaction completion is indicated by TLC. Themixture is cooled to room temperature and the solid which forms iscollected by vacuum filtration, washed with ethanol and dried to givethe product. If a precipitate does not form upon cooling of the reactionmixture, the mixture is concentrated and purified by columnchromatography.

C. Examples of Oxindole Syntheses

The following examples of the synthesis of representative oxindoles isnot to be construed as limiting the scope of this invention in anymanner whatsoever. Alternate routes to the oxindoles shown as well asother oxindoles to be used to make the compounds of this invention willbecome apparent to those skilled in the art based on the followingdisclosures. Such syntheses and oxindoles are within the scope andspirit of this invention.

5-Amino-2-oxindole

5-Nitro-2-oxindole (6.3 g) was hydrogenated in methanol over 10%palladium on carbon to give 3.0 g (60% yield) of the title compound as awhite solid.

5-Bromo-2-oxindole

2-Oxindole (1.3 g) in 20 mL acetonitrile was cooled to −10° C. and 2.0 gN-bromosuccinimide was slowly added with stirring. The reaction wasstirred for 1 hour at −10° C. and 2 hours at 0° C. The precipitate wascollected, washed with water and dried to give 1.9 g (90 % yield) of thetitle compound.

4-Methyl-2-oxindole

Diethyl oxalate (30 mL) in 20 mL of dry ether was added with stirring to19 g of potassium ethoxide suspended in 50 mL of dry ether. The mixturewas cooled in an ice bath and 20 mL of 3-nitro-6-xylene in 20 mL of dryether was slowly added. The thick dark red mixture was heated to refluxfor 0.5 hr, concentrated to a dark red solid, and treated with 10%sodium hydroxide until almost all of the solid dissolved. The dark redmixture was treated with 30% hydrogen peroxide until the red colorchanged to yellow. The mixture was treated alternately with 10% sodiumhydroxide and 30% hydrogen peroxide until the dark red color was nolonger present. The solid was filtered off and the filtrate acidifiedwith 6N hydrochloric acid. The resulting precipitate was collected byvacuum filtration, washed with water, and dried under vacuum to give 9.8g (45% yield) of 2-methyl-6-nitrophenylacetic acid as an off-whitesolid. The solid was hydrogenated in methanol over 10% palladium oncarbon to give 9.04. g of the title compound as a white solid.

7-Bromo-5-chloro-2-oxindole

5-Chloro-2-oxindole (16.8 g) and 19.6 g of N-bromosuccinimide weresuspended in 140 mL of acetonitrile and refluxed for 3 hours. Thin layerchromatography (silica, ethyl acetate) at 2 hours of reflux showed5-chloro-2-oxindole or N-bromosuccinimide (Rf 0.8), product (Rf 0.85)and a second product (Rf 0.9) whose proportions did not change afteranother hour of reflux. The mixture was cooled to 10° C., theprecipitate was collected by vacuum filtration, washed with 25 mL ofethanol and sucked dry for 20 minutes in the funnel to give 14.1 g ofwet product (56% yield). The solid was suspended in 200 mL of denaturedethanol and slurry-washed by stirring and refluxing for 10 minutes. Themixture was cooled in an ice bath to 10° C. The solid product wascollected by vacuum filtration, washed with 25 mL of ethanol and driedunder vacuum at 40° C. to give 12.7 g (51% yield) of7-bromo-5-chloro-2-oxindole.

5-Fluoro-2-oxindole

5-Fluoroisatin (8.2 g) was dissolved in 50 mL of hydrazine hydrate andrefluxed for 1.0 hr. The reaction mixtures were then poured in icewater. The precipitate was then filtered, washed with water and dried ina vacuum oven to afford the title compound.

5-Nitro-2-oxindole

2-Oxindole (6.5 g) was dissolved in 25 mL concentrated sulfuric acid andthe mixture maintained at −10 to −15° C. while 2.1 mL of fuming nitricacid was added dropwise. After the addition of the nitric acid thereaction mixture was stirred at 0° C. for 0.5 hr and poured intoice-water. The precipitate was collected by filtration, washed withwater and crystallized from 50% acetic acid. The crystalline product wasthen filtered, washed with water and dried under vacuum to give 6.3 g(70%) of 5-nitro-2-oxindole.

5-Aminosulfonyl-2-oxindole

To a 100 mL flask charged with 27 mL of chlorosulfonic acid was addedslowly 13.3 g of 2-oxindole. The reaction temperature was maintainedbelow 30° C. during the addition. After the addition, the reactionmixture was stirred at room temperature for 1.5 hr, heated to 68° C. for1 hr, cooled, and poured into water. The precipitate was washed withwater and dried in a vacuum oven to give 11.0 g of5-chlorosulfonyl-2-oxindole (50% yield) which was used without furtherpurification.

5-chlorosulfonyl-2-oxindole (2.1 g) was added to 10 mL of ammoniumhydroxide in 10 mL of ethanol and stirred at room temperature overnight.The mixture was concentrated and the solid collected by vacuumfiltration to give 0.4 g (20% yield) of the title compound as anoff-white solid.

5-Isopropylaminosulfonyl-2-oxindole

To a 100 mL flask charged with 27 mL chlorosulfonic acid was slowlyadded 13.3 g 2-oxindole. The reaction temperature was maintained below30° C. during the addition. The reaction mixture was stirred at roomtemperature for 1.5 hour, heated to 68° C. for 1 hour, cooled, andpoured into water. The precipitate which formed was filtered, washedwith water and dried in a vacuum oven to give 11.0 g (50%) of5-chlorosulfonyl-2-oxindole which was used without further purification.

A suspension of 3 g 5-chlorosulfonyl-2-oxindole, 1.15 g isopropylamineand 1.2 mL of pyridine in 50 mL of dichloromethane was stirred at roomtemperature for 4 hours during which time a white solid formed. Thesolid was collected by vacuum filtration, slurry-washed with hotethanol, cooled, collected by vacuum filtration and dried under vacuumat 40° C. overnight to give 1.5 g (45%) of5-isopropylaminosulfonyl-2-oxindole.

¹HNMR (360 MHz, DMSO-d6) δ 10.69 (s, br, 1H, NH), 7.63 (dd, J=2 and 8Hz, 1H), 7.59 (d, J=2 Hz, 1H), 7.32 (d, J=7 Hz, 1H, NH—SO₂—), 6.93 (d, J=8 Hz, 1H), 3.57 (s, 2H), 3.14-3.23 (m, 1H, CH—(CH₃)₂), 0.94 (d, J=7 Hz,6H, 2×CH₃).

5-Phenylaminosulfonyl-2-oxindole

A suspension of 5-chlorosulfonyl-2-oxindole (1.62 g, 7 mmol), aniline(0.782 mL, 8.4 mmol) and pyridine (1 mL) in dichloromethane (20 ml) wasstirred at room temperature for 4 hours. The reaction mixture wasdiluted with ethyl acetate (300 mL) and acidified with 1N hydrochloricacid (16 mL). The organic layer was washed with sodium bicarbonate andbrine, dried and concentrated. The residue was washed with ethanol (3mL) and then chromatographed on silica gel eluting withmethanol/dichloromethane 1:9 to give of5-phenylaminosulfonyl-2-oxindole.

¹HNMR (360 MHz, DMSO-d6) δ 10.71 (s, br, 1H, NH), 10.10 (s, br, 1H, NH),7.57-7.61 (m, 2H), 7.17-7.22 (m, 2H), 7.06-7.09 (m, 2H), 6.97-7.0 (m,1H), 6.88 (d, J=8.4 Hz, 1H), 3.52 (s, 2H).

2-Oxo-2,3-dihydro-1H-indole-5-sulfonic acid pyridin-3-ylamide

A solution of 5-chlorosufonyl-2-oxindole (3 g) and 3-aminopyridine (1.46g) in pyridine (15 mL) was stirred at room temperature overnight atwhich time a brown solid was present. The solid was filtered, washedwith ethanol and dried under vacuum to yield 1.4 g (38%) of2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid pyridin-3-ylamide.

¹HNMR (360 MHz, DMSO-d6) δ 10.74 (s, 1H, NH), 10.39 (s, 1H, SO₂NH),8.27-8.28 (d, 1H), 8.21-8.23 (m, 1H), 7.59-7.62 (m, 2H), 7.44-7.68 (m,1H), 7.24-7.28 (m, 1H), 6.69-6.71 (d, 1H), 3.54 (s, 2H).

MS m/z (APCI+) 290.2.

5-Phenyloxindole

5-Bromo-2-oxindole (5 g, 23.5 mmol) was dissolved in 110 mL toluene and110 mL ethanol with stirring and a little heat.Tetrakis(triphenylphosphine)palladium,(0) (1.9 g, 1.6 mmol) was addedfollowed by 40 mL (80 mmol) 2M aqueous sodium carbonate. To this mixturewas added benzene boronic acid (3.7 g, 30.6 mmol) and the mixture washeated in a 100° C. oil bath for 12 hours. The reaction was cooled,diluted with ethyl acetate (500 mL), washed with saturated sodiumbicarbonate (200 mL), water (200 mL), 1N HCl (200 mL) and brine (200mL). The organic layer was dried over magnesium sulfate and concentratedto afford a brown solid. Trituration with dichloromethane afforded 3.8 g(77%) of 5-phenyl-2-oxindole as a tan solid.

¹H NMR (360 MHz, DMSO-d6) δ 10.4 (br s, 1H, NH), 7.57 (dd, J=1.8 and 7.2Hz, 1H), 7.5 to 7.35 (m, 5H), 7.29 (m, 1H), 6.89 (d, J=8.2 Hz, 1H), 3.51(s, 2H, CH₂CO).

MS m/z 209 [M⁺].

In similar fashion, the following oxindoles can be prepared:

6-(3,5-Dichlorophenyl)-1,3-dihydroindol-2-one

¹H NMR (360 MHz, DMSO-d6) δ 10.46 (br, 1H, NH), 7.64 (d, J=1.8 Hz, 2H),7.57 (m, 1H), 7.27 (m, 2H), 7.05 (d, J=1.1 Hz, 1H), 3.5 (s, 2H).

MS-EI m/z 277/279 [M]⁺.

6-(4-Butylphenyl)-1,3-dihydroindol-2-one

¹H NMR (360 MHz, DMSO-d6) δ 10.39 (s, 1H, NH), 7.49 (d, J=8.0 Hz, 2H),7.25 (d, J=8 Hz, 3H), 7.17 (dd, J=1.5 and 7.8 Hz, 1H), 6.99 (d, J=1.5Hz, 1H), 3.48 (s, 2H, CH₂CO), 2.60 (t, J=7.5 Hz, 2 Hz, CH₂CH₃), 1.57 (m,2H, CH₂), 1.32 (m, 2H, CH₂), 0.9 (t, J=7.5 Hz, 3H, CH₃).

6-(5-Isopropyl-2-methoxyphenyl)-1,3-dihydroindol-2-one

¹H NMR (360 MHz, DMSO-d6) δ 10.29 (br s, 1H, NH), 7.16-7.21 (m, 2H),7.08 (d, J=2.4 Hz, 1H), 6.97-7.01 (m, 2H), 6.89 (d, J=0.8 Hz, 1H), 3.71(s, 3H, OCH₃), 3.47 (s, 2H, CH₂CO), 2.86 (m, 1H, CH(CH₃)₂), 1.19 (d,J=6.8 Hz, 6H, CH(CH₃)₂).

MS-EI m/z 281 [M]⁺.

6-(4-Ethylphenyl)-1,3-dihydroindol-2-one

¹H NMR (360 MHz, DMSO-d6) δ 10.39 (br s, 1H, NH), 7.50 (d, J=8.2 Hz,2H), 7.28 (d, J=8.2 Hz, 2H), 7.25 (d, J=7.5 Hz, 1H), 7.17 (dd, J=1.6 &7.5 Hz, 1H), 6.99 (d, J=1.6 Hz, 1H), 3.48 (s, 2H, CH₂CO), 2.63 (q, J=7.6Hz, 2H, CH₂CH₃), 1.20 (t, J=7.6 Hz, 3H, CH₂CH₃).

MS-EI m/z 237 [M]⁺.

6-(3-Isopropylphenyl)-1,3-dihydroindol-2-one

¹H NMR (360 MHz, DMSO-d6) δ 10.37 (br s, 1H, NH), 7.43 (m, 1H),7.35-7.39 (m, 1H), 7.17-7.27 (m, 3H), 7.01 (d, J=1.8 Hz, 1H), 3.49 (s,2H, CH₂CO), 2.95 (m, 1H, CH(CH₃)₂), 1.24 (d, J=6.8 Hz, 6H, CH(CH₃)₂).

MS-EI m/z 251 [M]⁺.

6-(2,4-Diethoxyphenyl)-1,3-dihydroindol-2-one

¹H NMR (360 MHz, DMSO-d6) δ 10.28, (br s, 1H, NH), 7.17 (m, 2H), 6.93(dd, J=1.6 & 7.6 Hz, 1H), 6.86 (d, J=1.6 Hz, 1H), 6.63 (d, J=2.4 Hz,1H), 6.58 (dd, J=2.4 & 8.5 Hz, 1H), 3.79 (s, 3H, OCH₃), 3.74 (s, 3H,OCH₃), 3.45 (s, 2H, CH₂CO).

MS-EI m/z 269 [M]⁺.

6-Pyridin-3-yl-1,3-dihydroindol-2-one

¹H NMR (360 MHz, DMSO-d6) δ 10.51 (s, 1H, NH), 8.81 (d, J=2.5 Hz, 1H),8.55 (dd, J=1.8 and 5.7 Hz, 1H), 8 (m, 1H), 7.45 (dd, J=5.7 and 9.3 Hz,1H), 7.3 (m, 2H), 7.05 (s, 1H), 3.51 (s, 2H, CH₂CO).

MS m/z 210 [M]⁺.

2-Oxo-2,3-dihydro-1H-indole-4-carboxylic acid(3-chloro-4-ethoxyphenyl)-amide

To a solution of 4-carboxy-2-oxindole (200 mg, 1.13 mmol) and3-chloro-4-methoxyphenylamine (178 mg, 1.13 mmol) in dimethylformamide(15 mL) at room temperature was addedbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP reagent, 997 mg, 2.26 mmol) followed by 4-dimethylaminopyridine(206 mg, 1.69 mmol). The mixture was stirred at room temperature for 72hours. The reaction was then diluted with ethyl acetate (300 mL), washedwith saturated sodium bicarbonate (100 mL), water, 2N hydrochloric acid(100 mL), water (3×200 mL) and brine. It was then dried over magnesiumsulfate and concentrated. The residue was triturated with ethyl acetateto give 2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid(3-chloro-4-methoxyphenyl)-amide as a pink solid.

¹HNMR (360 MHz, DMSO-d6) δ 10.50 (s, br, 1H, NH), 10.12 (s, br, 1H, NH),7.9 (s, J=2.5 Hz, 1H), 7.62 (dd, J=2.5 & 9 Hz, 1H), 7.38 (d, J=7.6 Hz,1H), 7.32 (t, J=7.6 Hz, 1H), 7.13 (d, J=9 Hz, 1H), 6.98 (d, J=7.6 Hz,1H), 3.83 (s, 3H, OCH₃), 3.69 (s, 2H, CH₂).

MS-EI m/z 316 [M]⁺.

4-Carboxy-2-oxindole

A solution of trimethylsilyldiazomethane in hexane (2 M) was addeddropwise to a solution of. 2.01 g 2-chloro-3-carboxy-nitrobenzene in 20mL methanol at room temperature until no further gas evolution occurred.Acetic acid was then added to quench excess trimethylsilyldiazomethane.The reaction mixture was evaporated under vacuum and the residue wasdried in an oven overnight. The 2-chloro-3-methoxycarbonylnitrobenzeneobtained was pure enough for the following reaction.

Dimethyl malonate (6.0 mL) was added to an ice-cold suspension of 2.1 gsodium hydride in 15 mL DMSO. The reaction mixture was stirred at 100°C. for 1 hour and then cooled to room temperature.2-Chloro-3-methoxycarbonylnitrobenzene (2.15 g) was added in one portionand the mixture was heated to 100° C. for 1.5 hours. The reactionmixture was then cooled to room temperature, poured into ice water,acidified to pH 5 and extracted with ethyl acetate. The organic layerwas washed with brine, dried over anhydrous sodium sulfate andconcentrated to give 3.0 g of the dimethyl2-methoxycarbonyl-6-nitrophenyl-malonate.

Dimethyl 2-methoxycarbonyl-6-nitrophenylmalonate (3.0 g) was refluxed in50 mL of 6 N hydrochloric acid overnight. The mixture was concentratedto dryness, 20 mL ethanol and 1.1 g of tin(II) chloride were added andthe mixture was refluxed for 2 hours. The mixture was filtered throughCelite, concentrated and chromatographed on silica gel using ethylacetate:hexane:acetic acid as eluent to give 0.65 g (37%) of4-carboxy-2-oxindole as a white solid.

¹HNMR (360 MHz, DMSO-d6) δ 12.96 (s, br, 1H, COOH), 10.74 (s, br, 1H,NH), 7.53 (d, J=8 Hz, 1H), 7.39 (t, J=8 Hz, 1H), 7.12 (d, J=8 Hz, 1H),3.67 (s, 2H).

D. Synthesis of Pyrrole Substituted 2-indolinones.

Example 14-Methyl-5-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylicacid

4-Methyl-2-pyrrolecarboxylic acid ethyl ester (commercially available)was formylated using method A to give (73%) of5-formyl-4-methyl-2-pyrrolecarboxylic acid ethyl ester. It was thenhydrolysed using method B to give5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid (58%).

Oxindole (133 mg, 1 mmol) was condensed with5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid (153 mg) using method Dto give 268 mg (100%) of the title compound as an orange-red solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.84 (s, br, 1H, NH), 12.84 (s, br, 1H,COOH), 10.98 (s, br, 1H, NH), 7.82 (d, J=7.5 Hz, 1H), 7.67 (s, 1H,H-vinyl), 7.18 (t, J=7.5 Hz, 1H), 7.01 (t, J=7.5 Hz, 1H), 6.88 (d, J=7.5Hz, 1H), 6.71 (d, J=2.2 Hz, 1H), 2.32 (s, 3H, CH₃).

MS (negative mode) 266.8 (M−1]⁺.

Example 24-Methyl-5-(1-methyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylicacid

1-Methyl-1,3-dihydroindol-2-one (147 mg, 1 mmol) was condensed with5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid (153 mg) using method Dto give 250 mg (86%) of the title compound.

¹HNMR (360 MHz, DMSO-d6) δ 13.82 (s, br, 1H, NH), 12.88 (s, br, 1H, 7.83(d, J=7.5 Hz, 1H), 7.65 (s, 1H, H-vinyl), 7.26 (t, J=7.5 Hz, 1H),7.02-7.09 (m, 2H), 6.70 (d, J=2.2 Hz, 1H), 2.32 (s, 3H, CH₃).

MS m/z 283.0 [M+1]⁺.

Example 34-Methyl-5-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylicacid methyl ester

Oxindole (105 mg, 0.79 mmol) was condensed with5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid methyl ester (110 mg,0.67 mmol) using method E to give 153.2 mg (81%) of the title compound.

¹HNMR (360 MHz, DMSO-d6) δ 13.98 (s, br, 1H, NH), 10.97 (s, br, 1H, NH),7.82 (d, J=7.6 Hz, 1H), 7.67 (s, 1H, H-vinyl), 7.2 (dt, J=1.2 & 7.7 Hz,1H), 7.01 (dt, J=1.2, 7.7 Hz, 1H), 6.90 (d, J=7.6. Hz, 1H), 6.77 (d, J=2Hz, 1H).

MS (ES) m/z 283 [M⁺+1].

Example 45-(5-Chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-methyl-1H-pyrrole-2-carboxylicacid ethyl ester

5-Chloro-1,3-dihydroindol-2-one (2.22 g, 13.2 mmol) was condensed with5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid ethyl ester (2.43 g)using method E to give 4.1 g (94%) of the title compound as an orangesolid.

¹HNMR (360 MHz, DMSO-d6) δ 13.95 (s, br, 1H, NH), 7.98 (d, J=2.2 Hz, 1H,H-4), 7.78 (s, 1H, H-vinyl), 7.18 (dd, J=2.2 & 8.3 Hz, 1H, H-6), 6.87(d, J=8.3 Hz, 1H, H-7), 7.34 (d, J=1.8 Hz, 1H, H-3′), 4.27 (q, J=7.2 Hz,2H, OCH₂CH₃), 2.33 (s, 3H, CH₃), 1.29 (t, J=7.2 Hz, 3H, OCH₂CH₃).

MS-EI m/z 330 [M⁺].

Example 55-(5-Chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-methyl-1H-pyrrole-2-carboxylicacid

A mixture of5-(5-chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-methyl-1H-pyrrole-2-carboxylicacid ethyl ester (1.3 g, 4 mmol) and potassium hydroxide in methanol (25mL) and ethanol (25 mL) was heated to reflux for overnight. Insolublematerials were removed by filtration and the mixture was neutralizedwith 6N hydrochloric acid to give 0.876 g (70%) of the title compound.

¹HNMR (360 MHz, DMSO-d6) δ 13.80 (s, br, 1H, NH), 12.90 (s, br, 1H,COOH), 11.06 (s, br, 1H, NH), 8.02 (d, J=1.8 Hz, 1H, H-4), 7.81 (s, 1H,H-vinyl), 7.20 (dd, J=1.8 & 8.3 Hz, 1H, H-6), 6.89 (d, J=8.3 Hz, 1H,H-7), 6.72 (d, J=1.8 Hz, 1H, H-3′), 2.35 (s, 3H, CH₃).

MS-EI m/z 302 [M⁺].

Example 65-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-methyl-1H-pyrrole-2-carboxylicacid (3-pyrrolidin-1-yl-propyl)amide

5-Bromo-1,3-dihydroindol-2-one (0.16 g, 0.76 mmol) was condensed with5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid(3-pyrrolidin-1-ylpropyl)amide (0.2 g, prepared by method C) to give 60mg (17%) of the title compound as an orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.61 (s, br, 1H, NH), 11.02 (s, br, 1H, NH),8.42 (t, J=5.8 Hz, 1H, CONHCH₂), 8.12 (d, J=1.8 Hz, 1H, H-4), 7.78 (s,1H, H-vinyl), 7.30 (dd, J=1.8 & 8.4 Hz, 1H, H-6), 6.82 (d, J=8.4 Hz, 1H,H-7), 6.77 (d, J=2.4 Hz, 1H, H-3′), 3.22-3.31 (m, 2H, CH₂), 2.38-2.43(m, 6H, 3×CH₂), 2.35 (s, 3H, CH₃), 1.62-1.71 (m, 6H, 3×CH₂).

MS-EI m/z 456 and 458 [M⁺−1 and M⁺+2].

Example 75-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-methyl-1H-pyrrole-2-carboxylicacid (3-diethylamino-propyl)amide

5-Bromo-1,3-dihydroindol-2-one (0.16 g, 0.75 mmol) was condensed with5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid(3-diethylaminopropyl)amide (0.2 g, prepared by method C) to give 30 mg(8%) of the title compound as an orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.61 (s, br, 1H, NH), 11.02 (s, br, 1H, NH),8.40 (m, 1H, CONHCH₂), 8.12 (d, J=1.5 Hz, 1H, H-4), 7.78 (s, 1H,H-vinyl), 7.30 (dd, J=1.5 & 8.2 Hz, 1H, H-6), 6.82 (d, J=8.2 Hz, 1H,H-7), 6.78 (d, J=2.4 Hz, 1H, H-3′), 3.23 (m, 2H, CH₂), 2.38-2.45 (m, 6H,CH₂ & N(CH₂CH₃)₂), 2.35 (s, 3H, CH₃), 1.61 (m, 2H, CH₂), 0.93 (t, J=7.1Hz, 6H, N(CH₂CH₃)₂).

MS-EI m/z 458 and 460 [M⁺−1 and M⁺+2].

Example 85-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylicacid (2-diethylaminoethyl)amide

5-Bromo-1,3-dihydroindol-2-one (212 mg, 1 mmol) was condensed with5-formyl-1H-pyrrole-2-carboxylic acid (2-diethylaminoethyl)amide(prepared from ethyl pyrrole-2-carboxylate by method A, B and then C) togive 162 mg (38%) of the title compound.

¹H NMR (300 MHz, DMSO-d6) δ 13.53 (s, br, 1H, NH), 11.06 (s, br, 1H,NH), 8.37 (t, 1H, CONHCH₂), 7.89 (m, 2H), 7.32 (dd, J=2.0 Hz, 1H), 6.96(s, 1H), 6.80-6.84 (m, 2H), 3.3 (m, 2H, CH₂), 2.45-2.55 (m, 6H,N(CH₂CH₃)₂ & CH₂), 0.95 (t, J=7.2 Hz, 6H, N(CH₂CH₃)₂).

MS-EI m/z 430 and 432 (M⁺−1 and M⁺+1].

Example 95-(2-Oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylicacid (2-diethylaminoethyl)amide

6-Phenyl-1,3-dihydroindol-2-one (209 mg, 1 mmol) was condensed with5-formyl-1H-pyrrole-2-carboxylic acid (2-diethylaminoethyl)amide to give182 mg (42%) of the title compound.

¹H NMR (300 MHz, DMSO-d6) δ 13.56 (s, br, 1H, NH), 11.06 (s, br, 1H,NH), 8.36 (t, 1H, CONHCH₂), 7.77 (s, 1H, H-vinyl), 7.73 (d, J=7.8 Hz,1H), 7.64 (d, J=7.2 Hz, 2H), 7.46 (m, 2H), 7.32 (m, 2H), 7.11 (s, 1H),6.96 (m, 1H), 6.80 (m, 1H), 3.31-3.32 (m, 2H, CH₂), 2.46-2.53 (m, 6H, N(CH₂CH₃) 2 & CH₂), 0.96 (t, J=6.9 Hz, 6H, N(CH₂CH₃)₂).

MS-EI m/z 428 [M⁺].

Example 105-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylicacid (2-diethylaminoethyl)-methyl-amide

5-Bromo-1,3-dihydroindol-2-one (212 mg, 1 mmol) was condensed with5-formyl-1H-pyrrole-2-carboxylic acid (2-diethylaminoethyl)methylamideto give 246 mg (55%) of the title compound.

¹H NMR (360 MHz, DMSO-d6) δ 13.54 (s, br, 1H, NH), 11.06 (s, br, 1H,NH), 7.90 (m, 2H), 7.33 (dd, J=1.8 & 8.4 Hz, 1H), 6.82-6.85 (m, 3H),3.55 (s, br, 2H, CH₂), 3.25 (s, br, 3H, NCH₃), 2.57 (t, J=6.5 Hz, 2H,CH₂), 2.45 (m, 4H, N(CH₂CH₃)₂), 0.91 (m, 6H, N(CH₂CH₃)₂).

MS-EI m/z 444 and 446 [M⁺−1 and M⁺+1].

Example 115-(2-Oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylicacid (2-diethylaminoethyl)methylamide

6-Phenyl-1,3-dihydroindol-2-one (209 mg, 1 mmol) was condensed with5-formyl-1H-pyrrole-2-carboxylic acid (2-diethylaminoethyl)methylamideto give 277 mg (63%) of the title compound.

¹H NMR (360 MHz, DMSO-d6) δ 13.58 (s, br, 1H, NH), 11.04 (s, br, 1H,NH), 7.78 (s, 1H, H-vinyl), 7.73 (d, J=7.8 Hz, 1H), 7.64 (d, J=7.5 Hz,2H), 7.46 (m, 2H), 7.33-7.36 (m, 2H), 7.11 (s, 1H), 6.84 (m, 1H), 6.78(m, 1H), 3.55 (s, br, 2H, CH₂), 3.25 (s, br, 3H, NCH₃), 2.58 (t, 2H,CH₂), 2.44 (m, 4H, N(CH₂CH₃)₂), 0.92 (m, 6H, N(CH₂CH₃)₂).

Example 123-Methyl-5-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylicacid (3-diethylaminopropyl)amide

Oxindole (66.5 mg, 0.5 mmol) was condensed with5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid(3-diethylaminopropyl)amide (prepared from3-formyl-3-methyl-1H-pyrrole-2-carboxylic acid ethyl ester by method Bthen C) to give 39 mg (21%) of the title compound.

¹H NMR (300 MHz, DMSO-d6) δ 13.34 (s, br, 1H, NH), 10.88 (s, br, 1H,NH), 7.62-7.67 (m, 3H), 7.17 (m, 1H), 6.99 (m, 1H), 6.87 (d, J=7.6 Hz,1H), 6.63 (d, J=1 Hz, 1H), 3.26-3.32 (m, 2H, CH₂), 2.41-2.48 (m, 6H, CH₂& N(CH₂CH₃)₂), 2.29 (s, 3H, CH₃), 1.63 (m, 2H, CH₂), 0.93 (t, J=7.2 Hz,6H, N(CH₂CH₃)₂).

MS-EI m/z 380 [M⁺].

Example 135-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (3-diethylamino-propyl)amide

5-Bromo-1,3-dihydroindol-2-one (106 mg, 0.5 mmol) was condensed with5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid(3-diethylaminopropyl)amide to give 35 mg (15%) of the title compound.

¹H NMR (300 MHz, DMSO-d6) δ 13.35 (s, br, 1H, NH), 11.00 (s, br, 1H,NH), 7.89 (d, J=1.9 Hz, 1H, H-4), 7.80 (s, 1H, H-vinyl), 7.74 (t, J=5.3Hz, 1H, CONHCH₂), 7.31 (dd, J=1.9 & 8.4 Hz, 1H, H-6), 6.83 (d, J=8.4 Hz,1H, H-7), 6.63 (s, 1H, H-3′), 3.26 (m, 2H, CH₂), 2.41-2.48 (m, 6H, CH₂ &N(CH₂CH₃)₂), 2.29 (s, 3H, CH₃), 1.63 (m, 2H, CH₂), 0.93 (t, J=7.1 Hz,6H, N(CH₂CH₃)₂).

MS-EI m/z 458 and 460 [M⁺−1 and M⁺+1];

Example 143-Methyl-5-(2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylicacid (3-diethylaminopropyl)amide

6-Phenyl-1,3-dihydroindol-2-one (105 mg, 0.5 mmol) was condensed with5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid(3-diethylaminopropyl)amide to give 67.8 (30%) of the title compound.

¹H NMR (300 MHz, DMSO-d6) δ 13.37 (s, br, 1H, NH), 11.02 (s, br, 1H,NH), 7.23-7.73 (m, 11H), 3.29 (m, 2H, CH₂), 2.41-2.48 (m, 6H, CH₂ &N(CH₂CH₃)₂), 2.29 (s, 3H, CH₃), 1.64 (m, 2H, CH₂), 0.94 (t, J=7.0 Hz,6H, N(CH₂CH₃)₂).

MS-EI m/z 456 [M⁺].

Example 155-(5-Methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (3-diethylamino-propyl)amide

5-Methoxy-1,3-dihydroindol-2-one (82.5 mg, 0.5 mmol) was condensed with5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid(3-diethylaminopropyl)amide to give 80 mg (39%) of the title compound.

¹H NMR (300 MHz, DMSO-d6) δ 13.45 (s, br, 1H, NH), 10.70 (s, br, 1H,NH), 7.68-7.70 (m, 2H), 7.32 (d, J=1.8 Hz, 1H), 6.72-6.79 (m, 2H), 6.60(s, 1H), 3.73 (s, 3H, OCH₃), 3.28 (m, 2H, CH₂), 2.41-2.48 (m, 6H, CH₂ &N(CH₂CH₃)₂), 2.29 (s, 3H, CH₃), 1.63 (m, 2H, CH₂), 0.93 (t, J=7.0 Hz,6H, N(CH₂CH₃)₂).

MS m/z 410 [M⁺].

Example 165-(6-Methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (3-diethylamino-propyl)amide

6-Methoxy-1,3-dihydroindol-2-one (82.5 mg, 0.5 mmol) was condensed with5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid(3-diethylaminopropyl)amide to give 63 mg (31%) of the title compound.

¹H NMR (300 MHz, DMSO-d6) δ 13.22 (s, br, 1H, NH), 10.86 (s, br, 1H,NH), 7.39-7.63 and 6.37-6.55 (m, 6H), 3.73 (s, 3H, OCH₃), 3.3 (m, 2H,CH₂), 2.45 (m, 6H, CH₂ & N(CH₂CH₃)₂), 2.28 (s, 3H, CH₃), 1.63 (m, 2H,CH₂), 0.93 (m, 6H, N(CH₂CH₃)₂).

MS m/z 410 [M⁺].

Example 173-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylicacid (2-diethylamino-ethyl)amide

4,5,6,7-Tetrahydro-2H-isoindole-1-carboxylic acid ethyl ester (May,Donald A.; Lash, Timothy D.; J. Org. Chem., 1992, 57:18, 4820-4828) wasformylated using method A then B to give3-formyl-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid.

5-Bromo-1,3-dihydroindol-2-one (1.43 g, 6.8 mmol) was condensed with3-formyl-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid(2-diethylaminoethyl)amide (1.97 g) to give 2.2 g (67%) of the titlecompound as a yellow-orange solid.

¹H NMR (360 MHz, DMSO-d6) δ 13.47 (s, 1H, NH), 11.0 (s, 1H, NH), 8.0 (d,1H, NH), 7.70 (s, 1H, CH), 7.28 (dd, J=2.1 and 8.2 Hz, 1H, ArH), 7.16(m, 1H, ArH), 6.8 (d, J=8.3 Hz, 1H, ArH), 3.3 (s, 2H, CONH), 2.5 (m, 6H,3×NCH₂), 2.78 (br m, 2H, pyrrole CH₂), 2.72 (br m, 2H, pyrroleCH₂), 1.7(br m, 4H, N(CH₂CH₃)₂), 1.74 (br s, 4H, CH₂CH₂CH₂CH₂), 0.96 (t, J=7.4Hz, 6H, N(CH₂CH₃)₂).

MS-EI m/z 484 and 486 (M⁺−1 and M⁺+1].

Example 183-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylicacid (3-diethylamino-propyl)amide

5-Bromo-1,3-dihydroindol-2-one (20 mg, 0.1 mmol) was condensed with3-formyl-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid(3-diethylaminopropyl)amide (30 mg) to give 33 mg (46%) of the titlecompound as an orange solid.

¹H NMR (360 MHz, DMSO-d6) δ 10.9 (s, 1H, NH), 8.0 (m, 1H, NH), 7.68 (m,1H, ArH), 7.4 (m, 1H, ArH), 7.29 (d, J=1.9 and 8.5 Hz, 1H, ArH), 6.8 (d,J=8 Hz, 1H, ArH), 2.7 (br m, 4H, 2×NCH₂), 2.4 (m, 8H, 4×NCH₂), 1.7 (brm, 4H, N(CH₂CH₃)₂), 1.6 (br m, 2H, CH₂CH₂CH₂), 0.93 (t, J=7.4 Hz, 6H,N(CH₂CH₃)₂).

MS-EI m/z 499 and 501 [M⁺ and M⁺+2].

Example 193-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylicacid (3-pyrrolidin-1-ylpropyl)amide

5-Bromo-1,3-dihydroindol-2-one (80 mg, 0.4 mmol) was condensed with3-formyl-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid(3-pyrrolidin-1-ylpropyl)amide (120 mg) to give 43 mg (22%) of the titlecompound as a tan-orange solid.

¹H NMR (360 MHz, DMSO-d6) δ 13.4 (s, 1H, NH), 10.9 (s, 1H, NH), 8.0 (m,1H, NH), 7.69 (m, 1H, ArH), 7.49 (m, 1H, ArH), 7.28 (d, J=1.7 and 7.8Hz, 1H, ArH), 6.8 (d, J=8 Hz, 1H, ArH), 3.3 (br m, 2H, 2×NCH₂), 2.8 (m,4H, 2×pyrroleCH₂), 2.5 (br m, 4H, N(CH₂CH₃)₂), 1.6 (br m, 8H,2×pyrroleCH₂CH₂, CH₂CH₂CH₂ and CONHCH₂).

MS-EI m/z 497 and 499 [M⁺ and M⁺+2].

Example 203-(2-Oxo-6-pyridin-3-yl-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylicacid (2-diethylaminoethyl)amide

6-Pyridin-3-yl-1,3dihydroindol-2-one (60 mg, 0.4 mmol) was condensedwith 3-formyl-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid(2-diethylaminoethyl)amide (80 mg) to give 50 mg (38%) of the titlecompound as a reddish solid.

¹H NMR (360 MHz, DMSO-d6) δ 13.4 (s, 1H, NH), 11 (s, 1H, NH), 8.9 (d,1H, NH), 8.7 (dd, 1H, ArH), 8.1 (dd, 1H, ArH), 7.9 (d, 1H, ArH), 7.6 (s,1H, CH), 7.5 (dd, 1H, ArH), 7.3 (dd, 1H, ArH), 7.1 (m, 2H, ArH), 3.35(m, 2H, CONHCH₂), 2.8 (m, 4H, 2×pyrroleCH₂), 2.5 (br m, 6H, N(CH₂CH₃)₂and NCH₂), 1.75 (br s, 4H, 2×pyrroleCH₂CH₂), 0.9 (t, 6H, N(CH₂CH₃)₂).

MS-EI m/z 484 [M⁺].

Example 214-Benzoyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (3-diethylaminopropyl)amide

To a mixture of benzoyl chloride (1 equiv.) and aluminum chloride (1equiv.) in dichloroethane at 0° C. was added ethyl3,5-dimethyl-2-pyrrolecarboxylate (1 equiv.). The mixture was stirred at80° C. for 4 hr. The mixture was then extracted with ethyl acetate(EtOAc) and H₂O. The combined organic extracts were washed withsaturated sodium bicarbonate and brine, dried and concentrated to give(51%) of 4-benzoyl-3,5-dimethyl-1H-pyrrole-2-carboxylic acid.

A mixture of 4-benzoyl-3,5-dimethyl-1H-pyrrole-2-carboxylic acid ethylester (4.13 g, 15.2 mmol) and ceric ammonium nitrate (33 g, 4 equiv.) in50 mL of tetrahydrofuran (THF):acetic acid (HOAc):H₂O 1:1:1 was refluxedovernight. The reaction mixture was then cooled, extracted with EtOAcand then basified to pH 9 with sodium carbonate. The organic layer wasthen washed with brine, dried (MgSO₄) and concentrated followed bycolumn chromatography to give 3.25 g (75%) of4-benzoyl-5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid ethyl ester asa yellow solid.

5-Bromo-1,3-dihydro-indol-2-one was condensed with4-benzoyl-5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid using method Dto give4-benzoyl-5-(5-bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid.

The above carboxylic acid was then coupled withN,N-diethyl-1,3-propanediamine using method C to give the titlecompound.

¹H NMR (360 MHz, DMSO-d6) δ 7.96 (m, 1H, CONHCH₂), 7.76 (d, J=7.0 Hz,2H), 7.68 (t, 1H), 7.56 (m, 2H), 7.40 (s, 2H) 7.33 (dd, J=1.6 & 8.3 Hz,1H, H-6), 6.84 (d, J=8.3 Hz, 1H, H-7), 3.33 (m, 2H, CH₂), 2.42-2.46 (m,6H, 3×CH₂), 2.10 (s, 3H, CH₃), 1.65 (m, 2H, CH₂), 0.94 (t, J=7.0 Hz, 6H,N(CH₂CH₃)₂).

MS Electron Impact m/z 564 [M⁺+1].

Example 224-Benzoyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (3-morpholin-4-ylpropyl)amide

¹H NMR (360 MHz, DMSO-d6) δ 14.10 (s, 1H, NH), 11.14 (br s, 1H, NH),7.92 (m, 1H, CONHCH₂), 7.75 (m, 2H), 7.69 (t, 1H), 7.56 (m, 2H), 7.42(m, 2H), 7.33 (dd, J=1.9 & 8.3 Hz, 1H, H-6), 6.85 (d, J=8.3 Hz, 1H,H-7), 3.56 (m, 4H, 2×CH₂), 3.33 (m, 2H, CH₂), 2.35 (m, 6H, 3×CH₂), 2.10(s, 3H, CH₃), 1.70 (m, 2H, CH₂).

Example 234-Benzoyl-3-methyl-5-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylicacid (3-pyrrolidin-1-ylpropyl)amide

¹H NMR (300 MHz, DMSO-d6) δ 14.18 (s, 1H, NH), 11.14 (br s, 1H, NH),8.01 (m, 1H, CONHCH₂), 7.74 (m, 1H), 7.67 (m, 1H), 7.55 (m, 1H), 7.32(s, 1H, H-vinyl), 7.17 (m, 1H), 6.92 (m, 1H), 3.36 (m, 2H, CH₂), 2.44(m, 6 H, 3×CH₂), 2.11 (s, 3H, CH₃), 1.65-1.75 (m, 6H, 3×CH₂).

MS Electron Impact m/z 482 [M⁺].

Example 244-Benzoyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (3-pyrrolidin-1-ylpropyl)amide

¹H NMR (360 MHz, DMSO-d6) δ 14.01 (s, 1H, NH), 11.18 (br s, 1H, NH),7.98 (m, 1H, CONHCH₂), 7.75 (m, 2H), 7.68 (m, 1H), 7.55 (m, 2H), 7.40(m, 2H), 7.33. (dd, J=2.0 & 8.2 Hz, 1H, H-6), 6.84 (d, J=8.2 Hz, 1H,H-7), 3.34 (m, 2H, CH₂), 2.42-2.47 (m, 6 H, 3×CH₂), 2.09 (s, 3H, CH₃),1.70 (m, 2H, CH₂), 1.64 (m, 4H, 2×CH₂).

Example 254-Benzoyl-3-methyl-5-(2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylicacid (3-pyrrolidin-1-ylpropyl)amide

¹H NMR (300 MHz, DMSO-d6) δ 14.15 (s, 1H, NH), 11.16 (br s, 1H, NH),7.98 (m, 1H, CONHCH₂), 7.77 (d, J=7.7 Hz, 2H), 7.69 (m, 1H), 7.53-7.63(m, 4H), 7.44 (m, 2H), 7.33-7.37 (m, 2H), 7.24 (s, 2H), 7.12 (s, 1H),3.36 (m, 2H, CH₂), 2.43-2.48 (m, 6 H, 3×CH₂), 2.12 (s, 3H, CH₃), 1.74(m, 2H, CH₂), 1.69 (m, 4H, 2×CH₂).

MS Electron Impact m/z 558 [M⁺].

Example 264-Benzoyl-5-(6-methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (3-pyrrolidin-1-ylpropyl)amide

¹H NMR (300 MHz, DMSO-d6) δ 13.99 (s, 1H, NH), 11.05 (br s, 1H, NH),7.93 (m, 1H, CONHCH₂), 7.72 (m, 2H), 7.65 (m, 1H), 7.54 (m, 2H), 7.15(s, 1H, H-vinyl), 7.04 (d, J=8.4 Hz, 1H, H-4), 6.51 (dd, J=2.3 & 8.4 Hz,1H, H-5), 6.44 (d, J=2.3 Hz, 1H, H-7), 3.74 (s, 3H, OCH₃), 3.35 (m, 2H,CH₂), 2.42-2.46 (m, 6 H, 3×CH₂), 2.10 (s, 3H, CH₃), 1.72 (m, 2H, CH₂),1.65 (m, 4H, 2×CH₂).

MS Electron Impact m/z 512 [M⁺].

Example 274-Benzoyl-5-(5-methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (3-pyrrolidin-1-ylpropyl)amide

¹H NMR (360 MHz, DMSO-d6) δ 14.24 (s, 1H, NH), 10.90 (br s, 1H, NH),7.97 (m, 1H, CONHCH₂), 7.75 (d, J=7.2 Hz, 2H), 7.69 (m, 1H), 7.56 (m,2H), 7.24 (s, 1H, H-vinyl), 6.79 (m, 2H), 6.66 (m, 1H), 3.67 (s, 3H,OCH₃), 3.34 (m, 2H, CH₂), 2.43-2.48 (m, 6 H, 3×CH₂), 2.14 (s, 3H, CH₃),1.71 (m, 2H, CH₂), 1.66 (m, 4H, 2×CH₂).

MS Electron Impact m/z 512 [M⁺].

Example 284-Benzoyl-5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (3-pyrrolidin-1-ylpropyl)amide

¹H NMR (300 MHz, DMSO-d6) δ 14.20 (s, 1H, NH), 11.14 (br s, 1H, NH),8.03 (m, 1H, CONHCH₂), 7.75 (m, 2H), 7.68 (m, 1H), 7.55 (m, 2H), 7.38(s, 1H, H-vinyl), 7.08 (m, 1H), 7.01 (m, 1H), 6.87 (m, 1H), 3.34 (m, 2H,CH₂), 2.42-2.48 (m, 6 H, 3×CH₂), 2.09 (s, 3H, CH₃), 1.70 (m, 2H, CH₂),1.65 (m, 4H, 2×CH₂).

MS Electron Impact m/z 500 [M⁺].

Example 294-Acetyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (3-diethylaminopropyl)amide

5-Bromo-1,3-dihydro-indol-2-one was condensed with4-acetyl-5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid(3-diethylaminopropyl)amide (prepared from4-acetyl-5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid ethyl ester bymethod B then C) to give the title compound.

¹H NMR (300 MHz, DMSO-d6) δ 14.19 (s, 1H, NH), 11.19 (br s, 1H, NH),8.15 (m, 1H, CONHCH₂), 8.11 (s, 1H, H-vinyl), 7.72 (d, J=1.8 Hz, 1H,H-4), 7.38 (dd, J=1.8 & 8.2 Hz, 1H, H-6), 6.87 (d, J=8.2 Hz, 1H, H-7),3.27 (m, 2H, CH₂), 2.57 (s, 3H, CH₃CO), 2.46 (m, 9 H, CH₃ & 3×CH₂), 1.64(m, 2H, CH₂), 0.93 (t, J=7.1 Hz, 6H, N(CH₂CH₃)₂).

Example 304-Acetyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (3-pyrrolidin-1-ylpropyl)amide

¹H NMR (300 MHz, DMSO-d6) δ 8.14 (m, 1H, CONHCH₂), 8.10 (s, 1H,H-vinyl), 7.70 (d, 1H, H-4), 7.36 (dd, J=1.6 & 8.1 Hz, 1H, H-6), 6.85(d, J=8.1 Hz, 1H, H-7), 3.32 (m, 2H, CH₂), 2.57 (s, 3H, CH₃CO), 2.44 (s,3H, CH₃), 2.35-2.48 (m, 6H, 3×CH₃), 1.65-1.71 (m, 6H, 3×CH₂).

MS m/z 499 & 501 [M⁺] & [M⁺+2].

Example 314-Acetyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (3-morpholin-4-ylpropyl)amide

¹H NMR (300 MHz, DMSO-d6) δ 14.20 (s, 1H, NH), 11.26 (br s, 1H, NH),8.09 (m, 2H, H-vinyl & CONHCH₂), 7.73 (d, J=1.5 Hz, 1H, H-4), 7.38 (dd,J=1.5 & 8.3 Hz, 1H, H-6), 6.87 (d, J=8.3 Hz, 1H, H-7), 3.55 (m, 4H,2×CH₂), 3.26 (m, 2H, CH₂), 2.57 (s, 3H, CH₃CO), 2.44 (s, 3H, CH₃), 2.35(m, 6H, 3×CH₃), 1.68 (m, 2H, CH₂).

MS-EI m/z 514 & 516 [M⁺−1] & [M⁺+1].

Example 324-Acetyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (3-hydroxypropyl)amide

¹H NMR (360 MHz, DMSO-d6) δ 14.17 (s, 1H, NH), 11.25 (br s, 1H, NH),8.10 (s, 1H, H-vinyl), 8.03 (m, 1H, CONHCH₂), 7.71 (br s, 1H, H-4), 7.37(br d, J=8.4 Hz, 1H, H-6), 6.87 (d, J=8.4 Hz, 1H, H-7), 4.51 (br s, 1H,OH), 3.51 (br s, 2H, CH₂), 3.36 (m, 2H, CH₂), 2.57 (s, 3H, CH₃CO), 2.43(s, 3H, CH₃), 1.70 (m, 2H, CH₂).

MS-EI m/z 445 & 447 [M⁺−1] & [M⁺+1].

Example 344-Acetyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (2-morpholin-4-ylethyl)amide

¹H NMR (360 MHz, DMSO-d6) δ 14.19 (s, 1H, NH), 11.14 (br s, 1H, NH),8.10 (.s, 1H, H-vinyl), 7.84 (m, 1H, CONHCH₂), 7.71 (d, J=1.8 Hz, 1H,H-4), 7.38 (dd, J=1.8 & 8.2 Hz, 1H, H-6), 6.87 (d, J=8.2 Hz, 1H, H-7),3.58 (m, 4H, 2×CH₂), 3.40 (m, 2H, CH₂), 2.57 (s, 3H, CH₃CO), 2.49 (m,4H, 2×CH₂), 2.45 (m, CH₃ & CH₂).

MS-EI m/z 500 & 502 [M⁺−1] & [M⁺+1].

Example 354-Acetyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (2-pyrrolidin-1-ylethyl)amide

¹H NMR (360 MHz, DMSO-d6) δ 14.17 (s, 1H, NH), 11.23 (s, 1H, NH), 8.11.(s, 1H, H-vinyl), 7.91 (m, 1H, CONHCH₂), 7.73 (d, J=1.9 Hz, 1H, H-4),7.39 (dd, J=1.9 & 8.3 Hz, 1H, H-6), 6.88 (d, J=8.3 Hz, 1H, H-7), 3.40(m, 2H, CH₂), 2.62 (m, 2H, CH₂), 2.57 (s, 3H, CH₃CO), 2.49 (m, 4H,2×CH₂), 2.44 (s, 3H, CH₃), 1.69 (m, 4H, 2×CH₂).

Example 364-Acetyl-5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylicacid (2-(4-hydroxyphenyl)ethyl]amide

¹H NMR (300 MHz, DMSO-d6) δ 14.21 (s, 1H, NH), 11.18 (s, 1H,OH), 9.09(s, 1H, NH), 8.06-8.10 (m, 2H), 7.73 (s, 1H), 7.38 (d, J=7.8 Hz, 1H),7.04 (d, J=7.1 Hz, 2H), 6.88 (d, J=7.8 Hz, 1H), 6.67 (d, J=7.1 Hz, 2H),3.42 (m, 2H, CH₂), 2.72 (m, 2H, CH₂), 2.56 (s, 3H, CH₃CO), 2.37 (s, 3H,CH₃).

MS-EI m/z 507 & 509 [M⁺−1] & [M⁺+1].

Example 375-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylicacid (3-diethylaminopropyl)amide

A mixture of 2-aminoacetophenone hydrochloride (1 equiv.), ethylisobutyrylacetate (1.2 equiv.) and sodium acetate (2.4 equiv.) in H₂Owas stirred at 100° C. for 18 hours and then cooled to room temperature.The aqueous layer was decanted off and the oil was dissolved in ethylacetate. It was then washed with water and brine and then dried to give(93%) of 2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylic acid ethyl esteras a red brown oil.

¹HNMR (300 MHz, DMSO-d6) δ 11.21 (s, br, 1H, NH), 7.14-7.27 (m, 5H),6.70 (d, J=2.7 Hz, 1H), 4.02 (q, J=7.1 Hz, 2H, OCH₂CH₃), 3.65 (m, 1H,CH(CH₃)₂), 1.22 (d, J=7.5 Hz, 6H, CH(CH₃)₂), 1.04 (t, J=7.1 Hz, 3H,OCH₂CH₃).

MS-EI m/z 257 [M⁺].

The above pyrrole was formylated using method A to give (41%)5-formyl-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylic acid ethyl esteras a reddish solid.

¹HNMR (300 MHz, DMSO-d6) δ 12.35 (s, br, 1H, NH), 9.14 (s, 1H, CHO),7.36 (s, 5H), 3.96 (q, J=7.1 Hz, 2H, OCH₂CH₃), 3.74 (m, 1H, CH(CH₃)₂),1.29 (d, J=6.9 Hz, 6H, CH(CH₃)₂), 0.90 (t, J=7.1 Hz, 3H, OCH₂CH₃).

MS-EI m/z 285 [M⁺].

The pyrrolecarboxylic acid ester was hydrolysed using method B to give(57%) of 5-formyl-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylic acid as abeige solid.

¹HNMR (300 MHz, DMSO-d6) δ 12.28 (s, br, 1H, COOH), 12.02 (s, br, 1H,NH), 9.10 (s, 1H, CHO), 7.35 (s, 5H), 3.81 (m, 1H, CH(CH₃)₂), 1.28 (d,J=6.9 Hz, 6H, CH(CH₃)₂).

MS-EI m/z 257 [M⁺].

5-Bromo-1,3-dihydroindol-2-one (120 mg, 0.31 mmol) was condensed with5-formyl-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylic acid(3-diethylaminopropyl)amide (prepared by method C) to give 120 mg (71%)of the title compound.

¹HNMR (300 MHz, DMSO-d6) δ 14.23 (s, br, 1H, NH), 11.08 (s, br, 1H, NH),7.38-7.55 (m, 7H, Ar—H & CONHCH₂), 7.30 (s, 1H, H-vinyl), 7.26 (dd,J=1.8& 7.8 Hz, 1H), 6.85 (d, J=8.7 Hz, 1H), 3.36 (m, 1H, CH(CH₃)₂), 3.07(m, 2H, CH₂), 2.34 (q, J=7.1 Hz, 4H, N(CH₂CH₃)₂), 2.22 (t, J=6.9 Hz, 2H,CH₂), 1.40 (m, 2H, CH₂), 1.31 (d, J=6.9 Hz, 6H, CH(CH₃)₂), 0.86 (t,J=7.1 Hz, 6H, N(CH₂CH₃)₂).

MS m/z 565.1 [M⁺+1].

Example 385-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylicacid (3-pyrrolidin-1-ylpropyl)amide

5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylicacid (127 mg, 0.28 mmol) was condensed with3-pyrrolidin-1-yl-propylamine (43 mg, 0.336 mmol) to give 140 mg (66%)of the title compound.

¹HNMR (300 MHz, DMSO-d6) δ 14.40 (s, br, 1H, NH), 7.38-7.47 (m, 7H),7.23-7.27 (m, 2H), 6.84 (d, J=8.1 Hz, 1H), 3.36 (m, 1H, CH(CH₃)₂), 3.08(m, 2H, CH₂), 2.30 (m, 4H, 2×CH₂), 2.20 (t, J=7.0 Hz, 2H, CH₂), 1.62 (m,4H, 2×CH₂), 1.42 (t, J=7.0 Hz, 2H, CH₂), 1.31 (d, J=7.2 Hz, 6H,CH(CH₃)₂).

MS-EI m/z 560 and 562 [M⁺−1 and M⁺+1].

Example 395-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylicacid (2-diethylaminoethyl)amide

5-Bromo-1,3-dihydroindol-2-one (57 ,g. 0.27 mmol) was condensed with5-formyl-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)amide (120 mg) to give 78 mg (53%) of the titlecompound as a yellow solid.

¹HNMR (300 MHz, DMSO-d6) δ 14.23 (s, br, 1H, NH), 11.09 (s, br, 1H, NH),7.38-7.51 (m, 6H), 7.25-7.28 (m, 2H), 7.19 (t, 1H, CONHCH₂), 6.85 (d,J=7.8 Hz,1H), 3.43 (m, 1H, CH(CH₃)₂), 3.11 (m, 2H, CH₂), 2.28-2.39 (m,6H, N(CH₂CH₃)₂ & CH₂, 1.31 (d, J=6.9 Hz, CH(CH₃)₂), 0.85 (t, J=7.0 Hz,6H, N(CH₂CH₃)₂.

MS-EI m/z 548 and 550 [M⁺−1 and M⁺+1].

Example 405-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylicacid [3-(4-methylpiperazin-1-yl)propyl]amide

5-Bromo-1,3-dihydroindol-2-one (53 mg, 0.25 mmol) was condensed with5-formyl-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylic acid[3-(4-methylpiperazin-1-yl)propyl]amide (300 mg) to give 65 mg of thetitle compound.

¹HNMR (300 MHz, DMSO-d6) δ 14.22 (s, br, 1H, NH), 11.08 (s, br, 1H, NH),7.23-7.50 (m, 9H), 6.85 (d, J=8.7 Hz, 1H), 3.37 (m, 1H, CH(CH₃)2), 3.05(m, 2H, CH₂), 2.24 (m, 8H, 4×CH₂), 2.11 (m, 5H, CH₂ & CH₃), 1.42 (m, 2H,CH₂), 1.31 (d, J=7.2 Hz, 6H, CH(CH₃)₂).

MS-EI m/z 589 and 591 [M⁺−1 and M⁺+1].

Example 415-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylicacid

5-Bromo-1,3-dihydroindol-2-one (170 mg, 0.8 mmol) was condensed with5-formyl-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylic acid (205 mg)using method D to give 210 mg (58%) of the title compound as a yellowsolid.

¹HNMR (300 MHz, DMSO-d6) δ 14.31 (s, br, 1H, NH), 11.16 (s, br, 1H, NH),7.26-7.44 (m, 7H), 7.11 (s, 1H, H-vinyl), 6.85 (d, J=7.8 Hz, 1H), 3.78(m, 1H, CH(CH₃)₂), 1.34 (d, J=6.9 Hz, 6H, CH(CH₃)₂).

MS-EI m/z 452 [M⁺+1].

Example 425-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-methyl-4-phenyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ylethyl)amide

5-Bromo-1,3-dihydroindol-2-one (44 mg, 0.21 mmol) was condensed with5-formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-ylethyl)amide (70 mg, prepared in the same manner as theisopropyl analog, above) to give 0.03 g (27%) of the title compound as ayellow solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.87 (s, br, 1H, NH), 11.11 (s, br, 1H, NH),7.36-7.51 (m, 6H), 7.26 (dd, J=1.8 & 8.1 Hz, 1H), 7.2 (s, 1H, H-vinyl),7.09 (m, 1H, CONHCH₂), 6.83 (d, J=8.1 Hz, 1H), 3.17 (m, 2H, NCH₂), 2.48(m, CH₃), 2.29-2.35 (m, 6H, 3×NCH₂), 1.59 (m, 4H, 2×CH₂).

MS-EI m/z 518 and 520 [M⁺−1 and M⁺+1].

Example 435-[6-(2-Methoxyphenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2-methyl-4-phenyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ylethyl)amide

6-(2-Methoxyphenyl)-1,3-dihydroindol-2-one (50 mg, 0.21 mmol) wascondensed with 5-formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-ylethyl)amide (70 mg) to give 0.04 g (35%) of the titlecompound as a yellow-orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.82 (s, br, 1H, NH), 11.02 (s, br, 1H, NH),7.48 (m, 2H), 7.43 (m, 1H), 7.38 (m, 2H), 7.32 (m, 1H), 7.24 (m, 2H),7.16 (s, 1H, H-vinyl), 7.08 (m, 2H), 7.03 (m, 1H), 7.0 (m, 2H), 3.74 (s,3H, OCH₃), 3.19 (m, 2H, NCH₂), 2.49 (m, CH₃), 2.32-2.38 (m, 6H, 3×NCH₂),1.59 (m, 4H, 2×CH₂).

MS-EI m/z 546 [M⁺].

Example 445-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-methyl-4-phenyl-1H-pyrrole-3-carboxylicacid (2-dimethylaminoethyl)amide

5-Bromo-1,3-dihydroindol-2-one (46 mg, 0.22 mmol) was condensed with5-formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid(2-dimethylaminoethyl)amide (65 mg) to give 60 mg (55%) of the titlecompound as a yellow solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.86 (s, br, 1H, NH), 11.09 (s, br, 1H, NH),7.47-7.49 (m, 2H), 7.38-7.41 (m, 4H), 7.26 (dd, J=2.2 & 8.3 Hz, 1H),7.21 (s, 1H, H-vinyl), 7.04 (m, 1H, CONHCH₂), 6.77 (d, J=8.3 Hz, 1H),3.15 (m, 2H, NCH₂), 2.48 (m, CH₃), 2.16 (t, J=6.8 Hz, 2H, 3×NCH₂), 2.02(s, 6H, 2×NCH₃).

MS m/z 493 and 494.8 [M⁺ and M⁺+2].

Example 455-[6-(2-Methoxyphenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2-methyl-4-phenyl-1H-pyrrole-3-carboxylicacid (2-dimethylaminoethyl)amide

6-(2-Methoxyphenyl)-1,3-dihydroindol-2-one (53 mg, 0.22 mmol) wascondensed with 5-formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid(2-dimethylaminoethyl)amide (65 mg) to give 0.05 g (44%) of the titlecompound as an orange gum.

¹HNMR (300 MHz, DMSO-d6) δ 13.82 (s, br, 1H, NH), 11.02 (s, br, 1H, NH),7.37-7.52 (m, 5H), 7.32 (m, 1H), 7.22-7.27 (m, 2H), 7.16 (s, 1H), 7.08(m, 2H), 7.03 (m, 1H), 7.0 (m, 2H), 3.74 (s, 3H, OCH₃), 3.15 (m, 2H,NCH₂), 2.49 (m, CH₃), 2.16 (t, J=6.5 Hz, 2H, NCH₂), 2.02 (s, 6H,2×NCH₃).

MS m/z 521 [M⁺+1].

Example 465-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-methyl-4-phenyl-1H-pyrrole-3-carboxylicacid ethyl ester

5-Bromo-1,3-dihydroindol-2-one (60 mg, 0.29 mmol) was condensed with5-formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid ethyl ester (75mg) to give 78 mg (60%) of the title compound as an orange solid.

¹HNMR (360 MHz, DMSO-d6) δ 14.01 (s, br, 1H, NH), 11.13 (s, br, 1H, NH),7.42-7.46 (m, 3H), 7.27-7.34 (m, 4H), 7.12 (s, 1H), 6.84 (dd, J=2.2 &8.3 Hz, 1H), 3.99-4.03 (m, 2H, OCH₂CH₃), 2.61 (s, 3H, CH₃), 0.98-1.03(m, 3H, OCH₂CH₃).

MS-EI m/z 450 and 452 [M⁺−1 and M⁺+1].

Example 475-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-methyl-4-phenyl-1H-pyrrole-3-carboxylicacid (3-diethylaminopropyl)amide

5-bromo-1,3-dihydroindol-2-one (0.47 g, 2.2 mmol) was condensed with5-formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid(3-diethylaminopropyl)amide (0.75 g) to give 0.11 g (42%) of the titlecompound as an orange solid.

¹HNMR (300MHz, DMSO-d6) δ 13.86 (s, br, 1H, NH), 7.42-7.46 (m, 3H),7.37-7.50 (m, 7H), 7.24-7.28 (m, 2H), 6.83 (d, J=8.1 Hz, 1H), 3.09 (m,2H, NCH₂), 2.45 (s, 3H, CH₃), 2.38 (q, J=7.1 Hz, 4H, 2×NCH₂CH₃), 2.26(t, J=6.9 Hz, 2H, NCH₂), 1.42 (m, 2H, NCH₂), 0.87 (t, J=7.1 Hz, 6H,2×NCH₂CH₃).

MS-EI m/z 535.0 and 537 [M⁺ and M⁺+2].

Example 485-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-dimethylamino-ethyl)amide

A mixture of tert-butyl 3-oxobutyrate and sodium nitrite (1 equiv.) inacetic acid was stirred at room temperature to givetert-butyl-2-hydroximino-3-oxobutyrate.

Ethyl-3-oxobutyrate (1 equiv.), zinc dust (3.8 equiv.) and the crudetert-butyl-2-hydroximino-3-oxobutyrate in acetic acid was stirred at 60°C. for 1 hr. The reaction mixture was poured into H₂O and the filtratewas collected to give (65%)2-tert-butyloxycarbonyl-3,5-dimethyl-4-ethoxycarbonylpyrrole.

A mixture of2-tert-butyloxycarbonyl-3,5-dimethyl-4-ethoxycarbonylpyrrole andtriethyl orthoformate (1.5 equiv.) in trifluoroacetic acid was stirredat 15° C. for 1 hour. The reaction was concentrated and the residue waspurified to give (64%) 2,4-dimethyl-3-ethoxycarbonyl-5-formylpyrrole asyellow needles.

2,4-Dimethyl-3-ethoxycarbonyl-5-formylpyrrole was hydrolyzed usingmethod B to give (90%) 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylicacid.

¹H NMR (360 MHz, DMSO-d6) δ 12 (br s, 2H, NH and CO₂H), 9.58 (s, 1H,CHO), 2.44 (s, 3H, CH₃), 2.40 (s, 3H, CH₃).

MS m/z 267 [M⁺].

5-Bromo-1,3-dihydroindol-2-one (0.17 g, 0.8 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-dimethylaminoethyl)amide (0.2 g, prepared by method C) using method Bto give 0.3 g (83%) of the title compound as a yellow solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.60 (s, br, 1H, NH), 10.94 (s, br, 1H, NH),8.07 (d, J=1.8 Hz, 1H, H-4), 7.75 (s, 1H, H-vinyl), 7.44 (t, J=5.2 Hz,1H, CONHCH₂), 7.24 (dd, J=1.8 & 8.4 Hz, 1H, H-6), 6.82 (d, J=8.4 Hz, 1H,H-7), 3.26-3.33 (m, 2H, NCH₂), 2.42 (s, 3H, CH₃), 2.41 (s, 3H, CH₃),2.38 (t, J=6.7 Hz, 2H, NCH₂), 2.18 (s, 6H, N(CH₃)₂).

MS-EI m/z 430, and 432 [M⁺−1 and M⁺+1].

Example 492,4-Dimethyl-5-(2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (2-dimethylaminoethyl)amide

6-Phenyl-1,3-dihydroindol-2-one (0.17 g, 0.8 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-dimethylaminoethyl)amide (0.2 g) to give 0.13 g (36%) of the titlecompound as a yellow-orange solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.59 (s, br, 1H, NH), 10.93 (, br, 1H, NH),7.85 (d, J=7.92 Hz, 1H, H-4), 7.63-7.65 (m, 3H), 7.40-7.47 (m, 3H,),7.32-7.36 (m, 1H, Ar—H), 7.30 (dd, J=1.6 & 7.9 Hz, 1H, H-5), 7.11 (d,J=1.6 Hz, 1H, H-7), 3.28-3.34 (m, 2H, NCH₂), 2.43 (s, 3H, CH₃), 2.41 (s,3H, CH₃), 2.38 (t, J=6.8 Hz, 2H, NCH₂), 2.18 (s, 6H, N(CH₃)₂).

MS-EI m/z 428 [M⁺].

Example 505-(5-Chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-dimethylamino-ethyl)amide

5-Chloro-1,3-dihydroindol-2-one (0.1 g, 0.6 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-dimethylaminoethyl)amide (0.15 g) to give 0.22 g (90%) of the titlecompound as a yellow solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.61 (s, br, 1H, NH), 10.98 (, br, 1H, NH),7.96 (d, J=2.0 Hz, 1H, H-4), 7.75 (s, 1H, H-vinyl), 7.50 (t, J=5.5 Hz,1H, CONHCH₂), 7.12 (dd, J=2.0 & 8.3 Hz, 1H, H-6), 6.86 (d, J=8.3 Hz, 1H,H-7), 3.26-3.31 (m, 2H, NCH₂), 2.42 (s, 3H, CH₃), 2.40 (s, 3H, CH₃),2.36 (t, J=6.6 Hz, 2H, NCH₂), 2.17 (s, 6H, N(CH₃)₂).

MS-EI m/z 386 [M⁺].

Example 515-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide

5-Bromo-1,3-dihydroindol-2-one (0.17 g, 0.8 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)amide (0.2 g) to give 0.09 g (26%) of the titlecompound as a yellow solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.61 (s, br, 1H, NH), 10.98 (, br, 1H, NH),8.09 (d, J=1.7 Hz, 1H, H-4), 7.76 (s, 1H, H-vinyl), 7.42 (t, J=5.5 Hz,1H, CONHCH₂), 7.24 (dd, J=1.7 & 8.0 Hz, 1H, H-6), 6.82 (d, J=8.0 Hz, 1H,H-7), 3.23-3.32 (m, 2H, NCH₂), 2.46-2.55 (m, 6H, 3×NCH₂), 2.43 (s, 3H,CH₃), 2.42 (s, 3H, CH₃), 0.96 (t, J=7.2 Hz, 6H, 2×NCH₂CH₃).

MS-EI m/z 458 and 460 [M⁺−1 and M⁺+1].

Example 525-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-yl-ethyl)amide

5-Bromo-1,3-dihydroindol-2-one (0.09 g, 0.4 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-ylethyl)amide (0.1 g) to give 0.14 g (81%) of the titlecompound as a yellow-orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.61 (s, br, 1H, NH), 10.98 (, br, 1H, NH),8.09 (d, J=1.9 Hz, 1H, H-4), 7.76 (s, 1H, H-vinyl), 7.53 (t, J=5.5 Hz,1H, CONHCH₂), 7.24 (dd, J=1.9 & 8.5 Hz, 1H, H-6), 6.81 (d, J=8.5 Hz, 1H,H-7), 3.29-3.35 (m, 2H, NCH₂), 2.54 (t, J=6.9 Hz, 2H, NCH₂), 2.47 (m,under DMSO), 2.42 (s, 3H, CH₃), 2.40 (s, 3H, CH₃), 1.66-1.69 (m, 4H,2×CH₂).

MS-EI m/z 456 and 458 [M⁺−1 and M⁺+1].

Example 535-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (3-imidazol-1-yl-propyl)amide

5-Bromo-1,3-dihydroindol-2-one (0.09 g, 0.4 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(3-imidazol-1-ylpropyl)amide (0.1 g) to give 0.1 g (59%) of the titlecompound as an orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.63 (s, br, 1H, NH), 10.99 (, br, 1H, NH),8.09 (d, J=2.2 Hz, 1H, H-4), 7.77 (s, 1H, H-vinyl), 7.71 (t, J=5.7 Hz,1H, CONHCH₂), 7.65 (s, 1H, Ar—H), 7.25 (dd, J=2.2 & 8.4 Hz, 1H, H-6),7.20 (s, 1H, Ar—H), 6.89 (s, 1H, Ar—H), 6.81 (d, J=8.4 Hz, 1H, H-7),4.02 (t, J=6.7 Hz, 2H, NCH₂), 3.18 (q, J=6.7 Hz, 2H, NCH₂), 2.43 (s, 3H,CH₃), 2.41 (s, 3H, CH₃), 1.93 (m, 2H, CH₂).

MS-EI m/z 467 and 469 [M⁺−1 and M⁺+1].

Example 545-[6-(2-Methoxyphenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-dimethylaminoethyl)amide

6-(2-Methoxyphenyl)-1,3-dihydroindol-2-one (30 mg, 0.13 mmol) wascondensed with 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-dimethylaminoethyl)amide (30 mg) to give 0.06 g (100%) of the titlecompound as a yellow-orange gum.

¹HNMR (300 MHz, DMSO-d6) δ 13.60 (s, br, 1H, NH), 10.89 (s, br, 1H, NH),7.79 (d, J=8.4 Hz, 1H), 7.63 (s, 1H, H-vinyl), 7.46 (t, J=5.5 Hz, 1H,CONHCH₂), 7.28-7.35 (m, 2H), 6.99-7.11 (m, 4H), 3.76 (s, 3H, OCH₃),3.27-3.31 (m, 2H, NCH₂), 2.43 (s, 3H, CH₃), 2.39 (s, 3H, CH₃), 2.37 (m,2H, NCH₂), 2.18 (s, 6H, N (CH₃)₂).

MS-EI m/z 458 [M⁺].

Example 555-[6-(3-Methoxyphenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-dimethylaminoethyl)amide

6-(3-Methoxyphenyl)-1,3-dihydroindol-2-one (30 mg, 0.13 mmol) wascondensed with 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-dimethylaminoethyl)amide (30 mg) to give 8 mg (14%) of the titlecompound as a yellow-orange solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.59 (s, br, 1H, NH), 10.92 (s, br, 1H, NH),7.84 (d, J=7.6 Hz, 1H), 7.65 (s, 1H, H-vinyl), 7.42 (m, 1H, CONHCH₂),7.36 (d, J=7.8 Hz, 1H), 7.29 (dd, J=1.6 & 7.6 Hz, 1H), 7.20 (d, J=7.8Hz, 1H), 7.14 (d, J=2.8 Hz, 1H), 7.11 (d, J=1.6 Hz, 1H), 6.91 (dd, J=2.8& 7.8 Hz, 1H), 3.82 (s, 3H, OCH₃), 3.21-3.33 (m, 2H, NCH₂), 2.43 (s, 3H,CH₃), 2.40 (s, 3H, CH₃), 2.36-2.40 (m, 2H, NCH₂), 2.18, (s, 6H,N(CH₃)₂).

MS-EI m/z 458 [M⁺].

Example 562,4-Dimethyl-5-(2-oxo-5-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (2-diethylaminoethyl)amide

5-Phenyl-1,3-dihydroindol-2-one (80 mg, 0.4 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)amide (0.1 g) using method B to give 79 mg (46%) ofthe title compound.

¹HNMR (300 MHz, DMSO-d6) δ 13.66 (s, br, 1H, NH), 10.95 (, br, 1H, NH),8.15 (d, J=1.2 Hz, 1H), 7.81 (s, 1H, H-vinyl), 7.71 (d, J=7.5 Hz, 1H),7.40-7.47 (m, 4H), 7.31 (m, 1H), 6.95 (d, J=8.1 Hz, 1H), 3.2-3.31 (m,2H, NCH₂), 2.46-2.55 (m, 6H, 3×NCH₂), 2.44 (s, 6H, 2×CH₃), 0.96 (t,J=7.4 Hz, 6H, 2×NCH₂CH₃).

MS-EI m/z 456 [M⁺].

Example 572,4-Dimethyl-5-(2-oxo-5-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ylethyl)amide

5-Phenyl-1,3-dihydroindol-2-one (0.04 g, 0.2 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-ylethyl)amide (0.04 g) to give the title compound as ayellow-orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.65 (s, br, 1H, NH), 10.96 (, br, 1H, NH),8.15 (d, J=1.0 Hz, 1H), 7.80 (s, 1H, H-vinyl), 7.71 (d, J=7.2 Hz, 2H),7.49 (t, J=6.3 Hz, 1H, CONHCH₂), 7.41-7.46 (m, 3H), 7.31 (m, 1H), 6.95(d, J=7.8 Hz, 1H), 4.08 (m, 4H, 2× NCH₂), 3.32 (m, 2H, NCH₂), 2.55. (t,J=7.1 Hz, 2H, NCH₂), 2.47 (m, under DMSO), 2.43 (s, 6H, 2×CH₃), 1.66 (m,4H, 2×CH₂).

MS-EI m/z 454 [M⁺].

Example 582,4-Dimethyl-5-(2-oxo-5-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (3-imidazol-1-ylpropyl)amide

5-Phenyl-1,3-dihydroindol-2-one (8 mg, 0.04 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(3-imidazol-1-ylpropyl)amide (10 mg) to give 10 mg (59%) of the titlecompound as an orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.67 (s, br, 1H, NH), 10.96 (, br, 1H, NH),8.16 (d, J=1.2 Hz, 1H), 7.81 (s, 1H, H-vinyl), 7.65-7.72 (m, 4H), 7.44(m, 3H), 7.31 (m, 1H, CONHCH₂), 7.21 (s, 1H, Ar—H), 4.02 (t, J=6.5 Hz,2H, NCH₂), 3.19 (q, J=6.5 Hz, 2H, CONHCH₂), 2.44 (s, 6H, 2×CH₃), 1.93(m, 2H, CH₂CH₂ CH₂).

MS-EI m/z 465 [M⁺].

Example 592,4-Dimethyl-5-(2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (2-diethylaminoethyl)amide

6-Phenyl-1,3-dihydroindol-2-one (0.08 g, 0.4 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)amide (0.1 g) to give 65 mg (38%) of the titlecompound as a yellow solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.61 (s, br, 1H, NH), 10.99 (, br, 1H, NH),7.86 (d, J=7.8 Hz, 1H), 7.62-7.66 (m, 3H), 7.40-7.47 (m, 3H), 7.28-7.36(m, 2H), 7.10 (d, J=1.2 Hz, 1H), 3.26 (m, 2H, NCH₂), 2.46-2.55 (m, 6H,3×NCH₂), 2.44 (s, 3H, CH₃), 2.41 (s, 3H, CH₃), 0.97 (t, J=7.2 Hz, 6H,2×NCH₂CH₃).

MS-EI m/z 456 [M⁺].

Example 602,4-Dimethyl-5-(2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ylethyl)amide

6-Phenyl-1,3-dihydroindol-2-one (30 mg, 0.15 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-ylethyl)amide (40 mg) to give 5.9 mg (8.5%) of the titlecompound as a yellow-orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.60 (s, br, 1H, NH), 10.99 (, br, 1H, NH),7.86 (d, J=7.8 Hz, 1H), 7.63-7.66 (m, 3H), 7.51 (m, 1H, CONHCH₂), 7.45(m, 2H), 7.28-7.36 (m, 2H), 7.10 (d, J=1.5 Hz, 1H), 3.31 (m, 6H,3×NCH₂), 2.55 (t, J=6.6 Hz, 2H, NCH₂), 2.43 (s, 3H, CH₃), 2.40 (s, 3H,CH₃), 1.67 (m, 4H, 2×CH₂).

MS-EI m/z 454 [M⁺].

Example 612,4-Dimethyl-5-(2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (3-imidazol-1-ylpropyl)amide

6-Phenyl-1,3-dihydroindol-2-one (8 mg, 0.04 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(3-imidazol-1-ylpropyl)amide (10 mg) to give 7.3 mg (43%) of the titlecompound as an orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.62 (s, br, 1H, NH), 10.99 (, br, 1H, NH),7.86 (d, J=8.2 Hz, 1H), 7.62-7.70 (m, 5H), 7.45 (m, 2H), 7.35 (m, 1H),7.30 (dd, J=1.4 & 8.2 Hz, 1H), 7.21 (s, 1H), 7.10 (d, J=1.4 Hz, 1H),6.89 (s, 1H), 4.02 (t, J=6.9 Hz, 2H, CH₂), 3.19 (m, 2H, NCH₂ CH₂), 2.43(s, 3H, CH₃), 2.41 (s, 3H, CH₃), 1.93 (t, J=6.9 Hz, 2H, NCH₂).

MS-EI m/z 465 [M⁺].

Example 625-[6-(3,5-Dichlorophenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylaminoethyl)amide

6-(3,5-Dichlorophenyl)-1,3-dihydroindol-2-one (64 mg, 0.23 mmol) wascondensed with 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)amide (60 mg) to give 53 mg (44%) of the titlecompound as a light brown solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.62 (s, br, 1H, NH), 10.99 (s, 1H, NH),7.89 (d, J=7.9 Hz, 1H, H-4), 7.69-7.71 (m, 3H), 7.55 (m, 1H, CONHCH₂),7.37 (m, 2H), 7.14 (d, J=1.4 Hz, 1H, H-7), 3.27 (m, 2H, NCH₂), 2.48-2.58(m, 6H, 3×NCH₂), 2.45 (s, 3H, CH₃), 2.42 (s, 3H, CH₃), 0.97 (t, J=6.8Hz, 6H, 3×NCH₂CH₃).

MS m/z 526.9 [M⁺+1].

Example 632,4-Dimethyl-5-(2-oxo-6-pyridin-3-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (2-diethylaminoethyl)amide

6-Pyridin-3-yl-1,3-dihydroindol-2-one (40 mg, 0.19 mmol) was condensedwith 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)amide (50 mg) give 29 mg (33%) of the titlecompound as a light orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.62 (s, br, 1H, NH), 11.05 (s, br, 1H, NH),8.86 (s, br, 1H), 8.53 (d, J=5.8 Hz, 1H), 8.04 (m, 1H), 7.91 (d, J=8.1Hz, 1H), 7.70 (s, 1H, H-vinyl), 7.40-7.48 (m, 2H), 7.35 (d, J=7.5 Hz,1H), 7.14 (s, 1H), 3.26 (m, 2H, NCH₂), 2.48-2.55 (m, 3×NCH₂), 2.42 (s,3H, CH₃), 2.38 (s, 3H, CH₃), 0.96 (t, J=6.9 Hz, 6H, 2×NCH₂CH₃).

MS-EI m/z 457 [M⁺].

Example 642,4-Dimethyl-5-(2-oxo-6-pyridin-3-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ylethyl)amide

6-Pyridin-3-yl-1,3-dihydroindol-2-one (60 mg, 0.28 mmol) was condensedwith 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-ylethyl)amide (75 mg) to give 90 mg (71%) of the titlecompound as a light orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.61 (s, br, 1H, NH), 11.05 (s, br, 1H, NH),8.86 (d, J=1.5 Hz, 1H), 8.54 (dd, J=1.5 & 4.8 Hz, 1H), 8.05 (m, 1H),7.91 (d, J=7.8 Hz, 1H), 7.70 (s, 1H, H-vinyl), 7.44-7.53 (m, 2H), 7.36(dd, J=1.5 & 8.1 Hz, 1H), 7.15 (d, J=1.2 Hz, 1H), 3.33 (m, 2H, NCH₂),2.47-2.57 (m, 6H, 3×NCH₂), 2.43 (s, 3H, CH₃), 2.41 (s, 3H, CH₃), 1.67(m, 4H, 2×CH₂).

MS-EI m/z 455 [M⁺].

Example 652,4-Dimethyl-5-(2-oxo-6-pyridin-3-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (3-dimethylaminopropyl)amide

6-Pyridin-3-yl-1,3-dihydroindol-2-one (42 mg, 0.2 mmol) was condensedwith 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(3-dimethylaminopropyl)amide (50 mg) to give 67 mg (75%) of the titlecompound as yellow-brown solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.61 (s, br, 1H, NH), 11.00 (s, br, 1H, NH),8.86 (s, br, 1H), 8.54 (s, br, 1H), 8.04 (m, 1H), 7.90 (d, J=8.0 Hz,1H), 7.69 (s, 1H, H-vinyl), 7.63 (m, 1H), 7.45-7.48 (m, 1H), 7.35 (dd,J=1.7 & 8.0 Hz, 1H), 7.15 (d, J=1.7 Hz, 1H), 3.21-3.27 (m, 2H, NCH₂),2.43 (s, 3H, CH₃), 2.41 (s, 3H, CH₃), 2.28 (m, 2H, NCH₂), 2.14 (s, 6H,2×NCH₃), 1.64 (m, 2H, CH₂).

MS-EI m/z 443 [M⁺].

Example 662,4-Dimethyl-5-(2-oxo-5-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (3-dimethylaminopropyl)amide

5-Phenyl-1,3-dihydroindol-2-one (67 mg, 0.32 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(3-dimethylaminopropyl)amide (81 mg) to give 40 mg (28%) of the titlecompound as an orange solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.66 (s, br, 1H, NH), 10.92 (s, br, 1H, NH),8.14 (s, 1H), 7.79 (s, 1H), 7.71 (m, 2H), 7.62 (m, 1H), 7.44 (m, 3H),7.32 (m, 1H), 6.95 (m, 1H), 3.33 (m, 2H, NCH₂), 2.43 (s, 6H, 2×CH₃),2.27 (m, 2H, NCH₂), 2.13 (s, 6H, 2×NCH₃), 1.63 (m, 2H, CH₂).

MS-EI m/z 442 [M⁺].

Example 672,4-Dimethyl-5-(2-oxo-5-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (3-diethylaminopropyl)amide

5-Phenyl-1,3-dihydroindol-2-one (1.5 g, 7.16 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(3-diethylaminopropyl)amide (2 g) to give 1.3 g (40%) of the titlecompound as a yellow-orange solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.64 (s, 1H, NH), 10.91 (s, 1H, NH), 8.14(d, J=1.4 Hz, 1H, ArH), 7.8 (s, 1H, ArH), 7.7 (dd, J=1.2 and 8.5 Hz, 2H,ArH), 7.6 (t, J=5.3 Hz, 1H, CONHCH₂), 7.4 (m, 3H, ArH), 7.3 (t, J=7.4Hz, 1H, ArH), 6.9 (d, J=8.0 Hz, 1H, ArH), 3.2 (m, 2H, CONHCH ₂), 2.5 (m,12H, 3×NCH ₂ and 2×CH ₃), 1.61 (m, 2H, CH₂CH ₂CH₂), 0.93 (t, J=6.7 Hz,6H, NCH₂CH ₃).

MS-EI m/z 470 [M⁺].

Example 682,4-Dimethyl-5-(2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (3-diethylaminopropyl)amide

6-Phenyl-1,3-dihydroindol-2-one (1.5 g, 7.16 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(3-diethylaminopropyl)amide (2 g) to give 1.9 g (57%) of the titlecompound as an orange solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.58 (s, 1H, NH), 10.94 (s, 1H, NH), 7.8 (d,J=7.9 Hz, 1H, ArH), 7.6 (m, 4H, ArH), 7.4 (t, J=7.5 Hz, 2H, ArH), 7.3(m, 2H), 7.1 (d, J=1.4 Hz, 1H, ArH), 3.2 (m, 2H, CONHCH₂), 2.5 (m, 12H,3×NCH₂and 2×CH₃), 1.61 (m, 2H, CH₂CH₂CH₂), 0.93 (t, J=6.7 Hz, 6H,NCH₂CH₃).

MS-EI m/z 470 [M⁺].

Example 693-[4-(3-Diethylaminopropylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-ylmethylene]-2-oxo-2,3-dihydro-1H-indole-4-carboxylicacid (3-chloro-4-methoxyphenyl)amide

2-Oxo-2,3-dihydro-1H-indole-4-carboxylic acid(3-chloro-4-methoxyphenyl)amide (1 g, 3.16 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(3-diethylaminopropyl)amide (1 g, 3.58 mmol) to give 1.7 g (85%) of thetitle compound as a yellow-orange solid.

MS-EI m/z 578.2 [M⁺].

Example 705-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (3-diethylamino-propyl)amide

5-Bromo-1,3-dihydroindol-2-one (0.5 g, 2.36 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(3-diethylaminopropyl)amide (0.51 g) to give 0.84 g of the titlecompound as a red-orange solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.61 (s, 1H, NH), 10.99 (s, 1H, NH), 8.09(d, J=1.8 Hz, 1H, ArH), 7.7 (m, 4H), 7.2 (dd, J=1.8 and 8.3 Hz, 2H,ArH), 6.8 (d, J=7.8 Hz, 1H, ArH), 3.3 (br s, 4H, 2×NCH₂), 3.2 (m, 2H,CONHCH₂), 2.6 (br s, 2H, NCH₂ and 2×CH₃), 2.4 (s, 6H, 2×CH₃), 1.66 (m,2H, CH₂CH₂CH₂), 0.98 (t, J=7.1 Hz, 6H, NCH₂CH₃).

MS-EI m/z 472 and 474 [M⁺−1 and M⁺+1].

Example 715-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-diisopropyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide

5-Bromo-1,3-dihydroindol-2-one (100 mg, 0.47 mmol) was condensed with5-formyl-2,4-diisopropyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)amide (150 mg) to give 0.15 g (62%) of the titlecompound as a yellow-orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.97 (s, 1H, NH), 10.95 (s, 1H, NH), 8.09(d, J=1.3 Hz, 1H, ArH), 7.84 (m, 1H), 7.79 (s, 1H), 7.23 (dd, J=1.3 and8.1 Hz, 1H, ArH), 6.8 (d, J=8.1 Hz, 1H, ArH), 3.5 (m, 1H, CH), 3.3 (m,3H, CH and NHCH₂), 2.5 (br m, 6H, 3×NCH₂), 1.28 (d, J=6.9 Hz, 6H,2×CH₃), 1.23 (d, J=6.6 Hz, 6H, 2×CH₃), 0.96 (m, 6H, 2×CH₂CH₃).

MS-EI m/z 514 and 516 [M⁺−1 and M⁺+1].

Example 725-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-diisopropyl-1H-pyrrole-3-carboxylicacid (3-diethylamino-propyl)amide

5-Bromo-1,3-dihydroindol-2-one (90 mg, 0.42 mmol) was condensed with5-formyl-2,4-diisopropyl-1H-pyrrole-3-carboxylic acid(3-diethylaminopropyl)amide (140 mg) to give 54 mg (25%) of the titlecompound as red-brown solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.98 (s, 1H, NH), 10.96 (s, 1H, NH), 8.09(d, J=1.7 Hz, 2H), 7.78 (s, 1H, H-vinyl), 7.23 (dd, J=1.7 and 8.1 Hz,1H, ArH), 6.82 (d, J=8.1 Hz, 1H, ArH), 3.5 (m, 1H, CH), 3.25 (m, 2H,NHCH₂), 3.15 (m, 1H, CH), 2.7 (br s, 6H, 3×NCH₂), 1.7 (br m, 2H,CH₂CH₂CH₂), 1.28 (d, J=6.9 Hz, 6H, 2×CH₃), 1.24 (d, J=5.9 Hz, 6H,2×CH₃), 1.06 (m, 6H, 2×CH₂CH₃).

MS-EI m/z 528 and 530 [M⁺−1 and M⁺+1].

Example 735-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-diisopropyl-1H-pyrrole-3-carboxylicacid (3-pyrrolidin-1-ylpropyl)amide

5-Bromo-1,3-dihydroindol-2-one (130 mg, 0.6 mmol) was condensed with5-formyl-2,4-diisopropyl-1H-pyrrole-3-carboxylic acid(3-pyrrolidin-1-ylpropyl)amide (150 mg, 0.45 mmol) to give 36 mg (15%)of the title compound as a tan-orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.98 (s, 1H, NH), 10.97 (s, 1H, NH), 8.10(d, J=1.6 Hz, 2H), 7.78 (s, 1H, H-vinyl), 7.23 (dd, J=1.6 and 7.6 Hz,1H, ArH), 6.82 (d, J=7.6 Hz, 1H, ArH), 3.5 (m, 1H, CH), 3.25 (m, 2H,NHCH₂), 3.15 (m, 1H, CH), 2.7 (br s, 6H, 3×NCH₂), 1.7 (br m, 6H,3×NCH₂CH₂), 1.28 (d, J=5.6 Hz, 6H, 2×CH₃), 1.24 (d, J=5.7 Hz, 6H,2×CH₃).

MS-EI m/z 526 and 528 [M⁺−1 and M⁺+1].

Example 745-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (pyridin-4-ylmethyl)-amide

5-Bromo-1,3-dihydroindol-2-one (170 mg, 0.8 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(pyridin-4-ylmethyl)amide (200 mg) to give 14 mg (4%) of the titlecompound as a yellow solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.67 (s, 1H, NH), 11.01 (s, br, 1H, NH),8.51 (dd, J=1.6 & 4.3 Hz, 2H), 8.23 (t, J=6.0 Hz, 1H, CONHCH₂), 8.11.(d, J=1.9 Hz, 1H), 7.78 (s, 1H, H-vinyl), 7.31 (d, J=6.0 Hz, 2H), 7.25(dd, J=1.9 & 8.1 Hz, 1H), 6.82 (d, J=8.1 Hz, 1H), 4.45 (d, J=6.0 Hz, 2H,NCH₂), 2.46 (s, 6H, 2×CH₃).

MS-EI m/z 450 and 452 [M⁺−1 and M⁺+1].

Example 755-(6-(4-Butylphenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ylethyl)amide

5-[6-(4-Butylphenyl)]-1,3-dihydroindol-2-one (50 mg, 0.19 mmol) wascondensed with 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-ylethyl)amide (50 mg) to give 74 mg (76%) of the titlecompound as an orange solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.58 (s, 1H, NH), 10.93 (s, br, 1H, NH),7.82 (d, J=7.9 Hz, 1H), 7.63 (s, 1H, H-vinyl), 7.54 (d, J=7.9 Hz, 2H),7.46 (m, 1H, CONH), 7.26 (m, 3H), 7.09 (s, 1H), 3.30 (m, 2H, CH₂),2.52-2.63. (m, 4H, 2×CH₂), 2.49 (m, 4H, 2×CH₂), 2.43 (s, 3H, CH₃), 2.40(s, 3H, CH₃), 1.68 (m, 4H, 2×CH₂), 1.58 (m, 2H, CH₂), 1.34 (m, 2H, CH₂),0.91 (t, J=7.2 Hz, 3H, CH₂CH₃).

MS-EI m/z 510 [M⁺].

Example 765-[6-(5-Isopropyl-2-methoxyphenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ylethyl)amide

6-(5-Isopropyl-2-methoxyphenyl)-1,3-dihydroindol-2-one (50 mg, 0.17mmol) was condensed with 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-yl-ethyl)amide (45 mg) to give 67 mg (75%) of thetitle compound as an orange solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.60 (s, 1H, NH), 10.82 (s, br, 1H, NH),7.77 (d, J=7.9 Hz, 1H), 7.61 (s, 1H, H-vinyl), 7.45 (m, 1H, CONH),7.0-7.19 (m, 5H), 3.73 (s, 3H, OCH₃), 3.32 (m, 2H, CH₂), 2.87 (m, 1H,CH(CH₃)₂), 2.56 (m, 2H, CH₂), 2.48 (m, 4H, 2×CH₂), 2.43 (s, 3H, CH₃),2.40 (s, 3H, CH₃), 1.68 (m, 4H, 2×CH₂), 1.21 (d, J=6.8 Hz, 6H,CH(CH₃)₂).

MS m/z 527.2 [M⁺+1].

Example 775-[6-(4-Ethylphenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ylethyl)amide

6-(4-Ethylphenyl)-1,3-dihydroindol-2-one (45 mg, 0.19 mmol) wascondensed 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-ylethyl)amide (50 mg) to give 60 mg. (65%) of the titlecompound as a yellow-orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.59 (s, 1H, NH), 10.96 (s, br, 1H, NH),7.83 (d, J=8.4 Hz, 1H), 7.64 (s, 1H, H-vinyl), 7.51-7.56 (m, 3H),7.25-7.30 (m, 3H), 7.08 (d, J=1 Hz, 1H), 3.31 (m, 2H, CH₂), 2.63 (m, 2H,CH₂CH₃), 2.55 (m, 2H, CH₂), 2.49 (m, 4H, 2×CH₂), 2.42 (s, 3H, CH₃), 2.40(s, 3H, CH₃), 1.67 (m, 4H, 2×CH₂), 1.20 (t, 7.5 Hz, 3H, CH₂CH₃).

MS-EI m/z 482 [M⁺].

Example 785-[6-(2,4-Dimethoxyphenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ylethyl)amide

6-(2,4-Dimethoxyphenyl)-1,3-dihydroindol-2-one (51 mg, 0.19 mmol) wascondensed with 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-ylethyl)amide (50 mg) to give 30 mg (31%) of the titlecompound as an orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.59 (s, 1H, NH), 10.86 (s, br, 1H, NH),7.75 (d, J=7.8 Hz, 1H), 7.60 (s, 1H, H-vinyl), 749 (m, 1H, CONH), 7.22(d, J=8.4 Hz, 1H), 7.03 (m, 1H), 6.97 (s, 1H), 6.58-6.65 (m, 2H), 3.79(s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.33 (m, 2H, CH₂), 2.55 (m, 2H, CH₂),2.50 (m, 4H, 2×CH₂), 2.42 (s, 3H, CH₃), 2.39 (s, 3H, CH₃), 1.67 (m, 4H,2×CH₂).

MS-EI m/z 514 [M⁺].

Example 795-[6-(3-Isopropylphenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ylethyl)amide

6-(3-Isopropylphenyl)-1,3-dihydroindol-2-one (48 mg, 0.19 mmol) wascondensed with 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-ylethyl)amide (50 mg) to give 59 mg (63%) of the titlecompound as an orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.63 (s, 1H, NH), 10.97 (s, br, 1H, NH),7.87 (d, J=7.8 Hz, 1H), 7.68 (s, 1H, H-vinyl), 7.24-7.55 (m, 6H), 7.13(s, 1H), 3.34 (m, 2H, CH₂), 3.30 (m, 1H, CH(CH₃)₂), 2.60 (m, 2H, CH₂),2.50 (m, 4H, 2×CH₂), 2.45 (s, 3H, CH₃), 2.43 (s, 3H, CH₃), 1.70 (m, 4H,2×CH₂), 1.27 (d, J=6.9 Hz, 6H, CH(CH₃)₂).

MS-EI m/z 496 [M⁺].

Example 805-(5-Fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide

5-Fluoro-1,3-dihydroindol-2-one (0.54 g, 3.8 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)amide to give 0.83 g (55%) of the title compound asa yellow green solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.66 (s, 1H, NH), 10.83 (s, br, 1H, NH),7.73 (dd, J=2.5 & 9.4 Hz, 1H), 7.69 (s, 1H, H-vinyl), 7.37 (t, 1H,CONHCH₂CH₂), 6.91 (m, 1H), 6.81-6.85 (m, 1H), 3.27 (m, 2H, CH₂), 2.51(m, 6H, 3×CH₂), 2.43 (s, 3H, CH₃), 2.41 (s, 3H, CH₃), 0.96 (t, J=6.9 Hz,6H, N(CH₂CH₃)₂).

MS-EI m/z 398 [M⁺].

Example 80 Alternative Synthesis5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide

Hydrazine hydrate (55%, 3000 mL) and 5-fluoroisatin (300 g) were heatedto 100° C. An additional 5-fluoro-isatin (500 g) was added in portions(100 g) over 120 minutes with stirring. The mixture was heated to 110°C. and stirred for 4 hours. The mixture was cooled to room temperatureand the solids collected by vacuum filtration to give crude(2-amino-5-fluoro-phenyl)-acetic acid hydrazide (748 g). The hydrazidewas suspended in water (700 mL) and the pH of the mixture adjusted to<pH 3 with 12 N hydrochloric acid. The mixture was stirred for 12 hoursat room temperature. The solids were collected by vacuum filtration andwashed twice with water. The product was dried under vacuum to give5-fluoro-1,3-dihydro-indol-2-one (600 g, 73% yield) as as a brownpowder. ¹H-NMR (dimethylsulfoxide-d₆) δ 3.46 (s, 2H, CH₂), 6.75, 6.95,7.05 (3×m, 3H, aromatic), 10.35 (s, 1H, NH).

MS m/z 152 [M+1].

3,5-Dimethyl-1H-pyrrole-2,4-dicarboxylic acid 2-tert-butyl ester 4-ethylester (2600 g) and ethanol (7800 mL) were stirred vigorously while 10 Nhydrochloric acid (3650 mL) was slowly added. The temperature increasedfrom25° C. to 35° C. and gas evolution began. The mixture was warmed to54° C. and stirred with further heating for one hour at which time thetemperature was 67° C. The mixture was cooled to 5° C. and 32 L of iceand water were slowly added with stirring. The solid was collected byvacuum filtration and washed three times with water. The solid was airdried to constant weight to give of 2,4-dimethyl-1H-pyrrole-3-carboxylicacid ethyl ester (1418 g, 87% yield) as a pinkish solid. ¹H-NMR(dimethylsulfoxide-d₆) δ 2.10, 2.35 (2×s, 2×3H, 2×CH₃), 4.13 (q, 2H,CH₂), 6.37 (s, 1H, CH), 10.85 (s, 1H, NH). MS m/z 167 [M+1].

Dimethylformamide (322 g) and dichloromethane (3700 mL) were cooled inan ice bath to 4° C. and phosphorus oxychloride (684 g) was added withstirring. Solid 2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethyl ester(670 g) was slowly added in aliquots over 15 minutes. The maximumtemperature reached was 18° C. The mixture was heated to reflux for onehour, cooled to 10° C. in an ice bath and 1.6 L of ice water was rapidlyadded with vigorous stirring. The temperature increased to 15° C. 10 NHydrochloric acid (1.6 L) was added with vigorous stirring. Thetemperature increased to 22° C. The mixture was allowed to stand for 30minutes and the layers allowed to separate. The temperature reached amaximum of 40° C. The aqueous layer was adjusted to pH 12-13 with 10 Npotassium hydroxide (3.8 L) at a rate that allowed the temperature toreach and remain at 55° C. during the addition. After the addition wascomplete the mixture was cooled to 10° C. and stirred for 1 hour. Thesolid was collected by vacuum filtration and washed four times withwater to give 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethylester (778 g, 100% yield) as a yellow solid. ¹H-NMR (DMSO-d₆) δ 1.25 (t,3H, CH₃), 2.44, 2.48 (2's, 2×3H, 2×CH₃), 4.16 (q, 2H, CH₂), 9.59 (s, 1H,CHO), 12.15 (br s, 1H, NH). MS m/z 195 [M+1].

5-Formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethyl ester (806 g),potassium hydroxide (548 g), water (2400 mL) and methanol (300 mL) wererefluxed for two hours with stirring and then cooled to 8° C. Themixture was extracted twice with dichloromethane. The aqueous layer wasadjusted to pH 4 with 1000 mL of 10 N hydrochloric acid keeping thetemperature under 15° C. Water was added to facilitate stirring. Thesolid was collected by vacuum filtration, washed three times with waterand dried under vacuum at 50° C. to give5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic (645 g, 93.5% yield) acidas a yellow solid. NMR (DMSO-d₆) δ 2.40, 2.43 (2×s, 2×3H, 2×CH₃), 9.57(s, 1H, CHO), 12.07 (br s, 2H, NH+COOH). MS m/z 168 [M+1].

5-Formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (1204 g) and 6020 mLof dimethylformamide were stirred at room temperature while1-(3-dimethyl-aminopropyl-3-ethylcarbodiimide hydrochloride (2071 g),hydroxybenzotriazole (1460 g), triethylamine (2016 mL) anddiethylethylenediamine (1215 mL) were added. The mixture was stirred for20 hours at room temperature. The mixture was diluted with 3000 mL ofwater, 2000 mL of brine and 3000 mL of saturated sodium bicarbonatesolution and the pH adjusted to greater than 10 with 10 N sodiumhydroxide. The mixture was extracted twice with 5000 mL each time of 10%methanol in dichloromethane and the extracts combined, dried overanhydrous magnesium sulfate and rotary evaporated to dryness. Themixture was with diluted with 1950 mL of toluene and rotary evaporatedagain to dryness. The residue was triturated with 3:1 hexane:diethylether (4000 mL). The solids were collected by vacuum filtration, washedtwice with 400 mL of ethyl acetate and dried under vacuum at 34° C. for21 hours to give 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylamino-ethyl)-amide (819 g, 43% yield) as a light brown solid.¹H-NMR (dimethylsulfoxide-d₆) δ 0.96 (t, 6H, 2×CH₃), 2.31, 2.38 (2×s,2×CH₃), 2.51 (m, 6H 3×CH₂), 3.28 (m, 2H, CH₂), 7.34 (m, 1H, amide NH),9.56 (s, 1H, CHO), 11.86 (s, 1H, pyrrole NH). MS m/z 266 [M+1].

5-Formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)-amide (809 g), 5-fluoro-1,3-dihydro-indol-2-one(438 g), ethanol (8000 mL) and pyrrolidine (13 mL) were heated at 78° C.for 3 hours. The mixture was cooled to room temperature and the solidscollected by vacuum filtration and washed with ethanol. The solids werestirred with ethanol (5900 mL) at 72° C. for 30 minutes. The mixture wascooled to room temperature. The solids were collected by vacuumfiltration, washed with ethanol and dried under vacuum at 54° C. for 130hours to give5-(5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)-amide (1013 g, 88% yield) as an orangesolid. ¹H-NMR (dimethylsulfoxide-d₆) δ 0.98 (t, 6H, 2×CH₃), 2.43, 2.44(2×s, 6H, 2×CH₃), 2.50 (m, 6H, 3×CH₂), 3.28 (q, 2H, CH₂), 6.84, 6.92,7.42, 7.71, 7.50 (5×m, 5H, aromatic, vinyl, CONH), 10.88 (s, 1H, CONH),13.68 (s, 1H, pyrrole NH). MS m/z 397 [M−1].

Example 813-[4-(2-Diethylaminoethylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-ylmethylene]-2-oxo-2,3-dihydro-1H-indole-6-carboxylicacid

2-oxo-2,3-dihydro-1H-indole-6-carboxylic acid (80 mg, 0.45 mmol) wascondensed with 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)amide to give 210 mg (92%) of the title compoundasayellow orange solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.6 (s, 1H, NH), 7.76 (d, J=8.0 Hz, 1H),7.66 (s, 1H, H-vinyl), 7.57 (dd, J=1.5 & 8.0 Hz, 1H), 7.40-7.42 (m, 2H),3.28 (m, 2H, CH₂), 2.88 (m, H-piperidine), 2.54 (m, 6H, 3×CH₂), 2.44 (s,3H, CH₃), 2.40 (s, 3H, CH₃), 1.56 (m, H-piperidine), 0.97 (t, J=6.98 Hz,6H, N(CH₂CH₃)₂).

MS m/z 424 [M⁺].

Example 825-(5-Dimethylsulfamoyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ylethyl)amide

2-Oxo-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide (90 mg, 0.38mmol) was condensed with 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ylethyl)amide (100 mg) to give 100 mg (54%) of thetitle compound as a yellow solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.65 (s, 1H, NH), 11.30 (s, br, 1H, NH),8.25 (d, 1H), 7.92 (s, 1H, H-vinyl), 7.48-7.53 (m, 2H), 7.07 (d, J=8.2Hz, 1H), 3.33 (m, 2H, CH₂), 2.61 (s, 6H, N(CH₃)₂), 2.56 (t, 2H, CH₂),2.49 (m, 4H, 2×CH₂), 2.45 (s, 3H, CH₃), 2.44 (s, 3H, CH₃), 1.67 (m, 4H,2×CH₂).

MS-EI m/z 485 [M⁺].

Example 835-[5-(3-Chlorophenylsulfamoyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ylethyl)amide

2-Oxo-2,3-dihydro-1H-indole-5-sulfonic acid (3-chloro-phenyl)amide (120mg, 0.38 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-ylethyl)amide (100 mg) to give 150 mg (69%) of the titlecompound as a yellow orange solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.55 (s, 1H, NH), 11.26 (br s, 1H, NH),10.30 (br s,1H, NH), 8.26 (d, 1H), 7.79 (s, 1H, H-vinyl), 7.51-7.57 (m,2H), 7.22 (t, J=8.1 Hz, 1H), 7.15 (m, 1H), 7.07 (m, 1H), 7.0 (m, 2H),3.44 (m, 2H, CH₂), 2.57 (t, J=7.0 Hz, 2H, CH₂), 2.49 (m, 4H, 2×CH₂),2.44 (s, 3H, CH₃), 2.43 (s, 3H, CH₃), 1.68 (m, 4H, 2×CH₂).

MS m/z 568 [M⁺].

Example 842,4-Dimethyl-5-(2-oxo-5-(pyridin-3-ylsulfamoyl)-1,2-dihydroindol-3-ylidenemethyl]-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ylethyl)amide

2-Oxo-2,3-dihydro-1H-indole-5-sulfonic acid pyridin-3-ylamide (110 mg,0.38 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-ylethyl)amide (100 mg) to give 150 mg (74%) of the titlecompound as an orange solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.58 (s, 1H, NH), 8.21 (d, J=2.0 Hz, 2H),8.04 (m, 1H), 7.76 (s, 1H, H-vinyl), 7.49-7.54 (m, 2H), 7.41 (m, 1H),7.14 (m, 1H), 6.94 (d, J=8.5 Hz, 1H), 3.33 (m, 2H, CH₂), 2.56 (t, J=7.06Hz, 2H, CH₂), 2.49 (m, 4H, 2×CH₂), 2.43 (s, 6H, 2×CH₃), 1.68 (m, 4H,2×CH₂).

MS m/z 535 [M⁺].

Example 853-(3,5-Dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrol-2-ylmethylene]-4-(2-hydroxyethyl)-1,3-dihydroindol-2-one

4-(2-Hydroxyethyl)-1,3-dihydroindol-2-one (71 mg, 0.4 mmol) wascondensed with3,5-dimethyl-4-(4-methyl-piperazine-1-carbonyl)-1H-pyrrole-2-carbaldehydeto give 90 mg (55%) of the title compound as an orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 14.25 (s, 1H, NH), 10.88 (s, 1H, NH), 7.57(s, 1H, H-vinyl), 7.03 (m, 1H), 6.75-6.82 (m, 2H), 4.86 (m, 1H, OH),3.70 (m, 2H, CH₂), 3.04 (m, 2H, CH₂), 2.48 (m, 4H, 2×CH₂), 2.28 (br s,7H), 2.19 (s, 3H, CH₃), 2.18 (s, 3H, CH₃).

MS m/z (+ve) 4.09.3 [M⁺].

Example 863-[3,5-Dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrol-2-ylmethylene]-2-oxo-2,3-dihydro-1H-indole-5-sulfonicacid phenylamide

2-Oxo-2,3-dihydro-1H-indole-5-sulfonic acid phenylamide (110 mg, 0.4mmol) was condensed with3,5-dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrole-2-carbaldehyde(100 mg) to give 50 mg (24%) of the title compound as a yellow solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.52 (s, 1H, NH), 11.26 (s, 1H, NH), 10.08(s, 1H, NH), 8.21 (d, J=1.6 Hz, 1H), 7.75 (s, 1H, H-vinyl), 7.50 (dd,J=1.6 & 8.3 Hz, 1H), 7.19 (m, 2H), 7.10 (m, 2H), 6.97 (m, 2H), 2.49 (m,4H, 2×CH₂), 2.28 (m, 10H, 2×CH₃ & 2×CH₂), 2.18 (s, 3H, CH₃).

MS-EI m/z 519 [M⁺].

Example 875-(5-Dimethylsulfamoyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylaminoethyl)amide

2-Oxo-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide (90 mg, 0.38mmol) was condensed with 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylaminoethyl)amide (100 mg) to give 80 mg (43%) of thetitle compound as a yellow solid.

¹HNMR (300 MHz, DMSO-d6) δ 11.30 (s, 1H, NH), 8.27 (d, J=1.7 Hz, 1H),7.94 (s, 1H, H-vinyl), 7.49 (dd, J=1.7 & 8.0 Hz, 1H), 7.44 (m, 1H,CONHCH₂CH₂), 7.07 (d, J=8.0 Hz, 1H), 3.26 (m, 2H, CH₂), 2.60 (s, 6H,N(CH₃)₂), 2.53. (m, 2H, CH₂), 2.45-2.50 (m, 10H, 2×CH₃ & N(CH₂CH₃)₂,0.96 (t, J=7.2 Hz, 6H, N(CH₂CH₃)₂).

MS-EI m/z 487 [M⁺].

Example 885-(5-(3-Chlorophenylsulfamoyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylaminoethyl)amide

2-Oxo-2,3-dihydro-1H-indole-5-sulfonic acid (3-chloro-phenyl)amide (120mg, 3.8 mmol) was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)amide (100 mg) to give 80 mg (37%) of the titlecompound as a yellow solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.55 (s, 1H, NH), 11.24 (s, 1H, NH), 10.29(s, 1H, NH), 8.25 (d, J=1.87 Hz, 1H), 7.79 (s, 1H, H-vinyl), 7.52 (dd,J=1.87 & 8.3 Hz, 1H), 7.42 (m, 1H, CONHCH₂CH₂), 7.22 (t, J=8.02 Hz, 1H),7.15 (t, J=2 Hz, 1H), 7.08 (m, 1H), 7.0 (m, 2H), 3.27 (m, 2H, CH₂),2.48-2.57 (m, 6H, 3×CH₂), 2.45 (s, 3H, CH₃), 2.44 (s, 3H, CH₃), 0.97 (t,J=7.0 Hz, 6H, N(CH₂CH₃)₂).

MS m/z 570.1 [M⁺].

Example 953-(2-Oxo-5-phenyl-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylicacid ethyl ester

¹HNMR (360 MHz, DMSO-d6) δ 13.74 (s, 1H, NH), 11.00 (s, 1H, NH), 8.13(d, J=1.7 Hz, 1H), 7.74 (s, 1H, H-vinyl), 7.70 (d, J=7.7 Hz, 2H), 7.49(dd, J=1.7 & 8.0 Hz, 1H), 7.44 (t, J=7.7 Hz, 2H), 7.32 (m, 1H), 6.96 (d,J=8.0 Hz, 1H), 4.26 (q, J=7.0 Hz, 2H, OCH₂CH₃), 2.79 (m, 2H, CH₂), 2.72(m, 2H, CH₂), 1.73 (m, 4H, 2×CH₂), 1.30 (t, J=7.0 Hz, 3H, OCH₂CH₃).

MS-EI m/z 412 [M⁺].

Example 993-(2-Oxo-5-phenylsulfamoyl-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylicacid ethyl ester

¹HNMR (360 MHz, DMSO-d6) δ 13.64 (s, 1H, NH), 11.33 (s, 1H, NH), 10.07(s, 1H, NH), 8.24 (d, J=1.8 Hz, 1H), 7.74 (s, 1H, H-vinyl), 7.57 (dd,J=1.8 & 8.0 Hz, 1H), 7.21 (t, J=7.6 Hz, 2H), 7.11 (d, J=7.6 Hz, 2H),6.99 (d, J=8.0 Hz, 1H), 6.98 (d, J=7.6 Hz, 1H), 4.27 (q, J=7.0 Hz, 2H,OCH₂CH₃), 2.80 (m, 2H, CH₂), 2.73 (m, 2H, CH₂), 1.73 (m, 4H, 2×CH₂),1.30 (t, J=7.0 Hz, 3H, OCH₂CH₃).

MS-EI m/z 491 [M⁺].

Example 1093-[3-(Morpholine-4-carbonyl)-4,5,6,7-tetrahydro-2H-isoindol-1-ylmethylene]-2-oxo-2,3-dihydro-1H-indole-6-carboxylicacid

¹HNMR (360 MHz, DMSO-d6) δ 13.60 (s, 1H, NH), 12.75 (br s, 1H, COOH),11.08 (s, 1H, NH), 7.85 (d, J=7.8 Hz, 1H), 7.71 (s, 1H, H-vinyl), 7.62(dd, J=1.4 & 7.8 Hz, 1H), 7.41 (d, J=1.4 Hz, 1H), 3.65 (m, 4H, 2×CH₂),3.55 (m, 4H, 2×CH₂), 2.81 (m, 2H, CH₂), 2.54 (m, 2H, CH₂) 1.73 (m, 4H,2×CH₂).

MS-EI m/z 421 [M⁺].

Example 1125-Bromo-3-(3-(pyrrolidine-1-carbonyl)-4,5,6,7-tetrahydro-2H-isoindol-1-ylmethylene]-1,3-dihydro-indol-2-one

¹HNMR (360 MHz, DMSO-d6) δ 13.56 (s, 1H, NH), 11.00 (s, 1H, NH), 8.05(d, J=1.8 Hz, 1H), 7.74 (s, 1H, H-vinyl), 7.28 (dd, J=1.3 & 8.3 Hz, 1H),6.83 (d, J=8.3 Hz, 1H), 3.57 (m, 4H, 2×CH₂), 2.79 (m, 2H, CH₂), 2.65 (m,2H, CH₂), 1.88 (m, 4H, 2×CH₂), 1.71 (m, 4H, 2×CH₂).

MS-EI m/z 439 & 441 [M⁺−1] & [M⁺+1].

Example 1143-(3-Dimethylcarbamoyl-4,5,6,7-tetrahydro-2H-isoindol-1-ylmethylene)-2-oxo-2,3-dihydro-1H-indole-6-carboxylicacid

¹HNMR (360 MHz, DMSO-d6) δ 13.60 (s, 1H, NH), 12.72 (br s, 1H, COOH),11.05 (s, 1H, NH), 7.85 (d, J=7.9 Hz, 1H), 7.72 (s, 1H, H-vinyl), 7.62(dd, J=1.3 & 7.9 Hz, 1H), 7.42 (d, J=1.3 Hz, 1H), 3.03 (s, 6H, N(CH₃)₂),2.81 (m, 2H, CH₂), 2.55 (m, 2H, CH₂), 1.73 (m, 4H, 2×CH₂).

MS-EI m/z 379 [M⁺].

Exapmle 1154-Methyl-5-(5-methylsulfamoyl-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid

¹HNMR (300 MHz, DMSO-d6) □ 13.56 (br s, 1H, NH), 8.24 (d, J=1.5 Hz, 1H),7.86 (s, 1H, H-vinyl), 7.74 (d, J=2.96 Hz, 1H), 7.56 (dd, J=1.5 & 8.1Hz, 1H), 7.20 (br m, 1H, NHCH₃), 7.03 (d, J=8.1 Hz, 1H), 2.57 (s, 3H,CH₃), 2.41 (s, 3H, CH₃).

MS-EI m/z 361 [M⁺].

Example 116([4-Methyl-5-(4-methyl-5-methylsulfamoyl-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carbonyl]-amino}-aceticacid ethyl ester

4-Methyl-1H-pyrrole-3-carboxylic acid ethyl ester (lit. ref. D. O.Cheng, T. L. Bowman and E. LeGoff; J. Heterocyclic Chem.; 1976; 13;1145-1147) was formylated using method A, hydrolysed using method Bfollowed by amidation (method C) to give[(5-formyl-4-methyl-1H-pyrrole-3-carbonyl)-amino]-acetic acid ethylester.

4-Methyl-5-methylaminosulfonyl-2-oxindole (50 mg, 0.21 mmol) wascondensed with [(5-formyl-4-methyl-1H-pyrrole-3-carbonyl)-amino]-aceticacid ethyl ester (100 mg, 0.42 mmol) and piperidine (0.1 mL) in ethanol(2 mL) to give 50 mg (52%) of the title compound.

¹HNMR (360 MHz, DMSO-d6) δ 13.59 (s, 1H, NH), 11.29 (v.br. s, 1H,NH—CO), 8.33 (t, J=5.8 Hz, 1H, CONHCH₂), 7.83 (d, J=3.11 Hz, 1H), 7.80(s, 1H, H-vinyl), 7.71 (d, J=8.5 Hz, 1H), 7.34 (br m, 1H, NHCH₃), 6.89(d, J=8.5 Hz, 1H), 4.11 (q, J=7.1 Hz, 2H, OCH₂CH₃), 3.92 (d, J=5.8 Hz,2H, GlyCH₂), 2.86 (s, 3H, CH₃), 2.48 (s, 3H, CH₃), 2.42 (d, J=4.71 Hz,3H, HNCH₃), 1.20 (t, J=7.1 Hz, 3H, OCH₂CH₃).

MS-EI m/z 460 [M⁺].

Example 117{[4-Methyl-5-(5-methylsulfamoyl-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carbonyl]-amino}-aceticacid ethyl ester

A mixture of 5-methylaminosulfonyl-2-oxindole (0.06 g, 0.22 mmol),[(5-formyl-4-methyl-1H-pyrrole-3-carbonyl)-amino]-acetic acid ethylester (0.075 g, 0.27 mmol) and piperidine (2 drops) in ethanol (5 mL)was heated in a sealed tube at 90° C. for 12 hrs. After cooling, theprecipitate was collected by vacuum filtration, washed with ethanol,triturated with dichloromethane/ether and dried to give 0.035 g (36%) ofthe title compound as a yellowish brown solid.

¹H NMR (360 MHz, DMSO-d6 ) δ 13.6 (s, 1H, NH), 11 (v.br. s, 1H, NH—CO),8.30 (t, J=5.7 Hz, 1H, CONHCH₂), 8.25 (d, J=1.2 Hz, 1H), 7.88 (s, 1H,H-vinyl), 7.84 (d, J=3.3 Hz, 1H), 7.57 (dd, J=1.9 & 8.5 Hz, 1H), 7.14(br m, 1H, NHCH₃), 7.04 (d, J=8.5 Hz, 1H), 4.11 (q, J=6.7 Hz, 2H,OCH₂CH₃), 3.92 (d, J=5.7 Hz, 2H, GlyCH₂), 2.55 (s, 3H, CH₃), 2.41 (m,3H, NCH₃), 1.20 (t, J=6.7 Hz, 3H, OCH₂CH₃).

MS m/z 446 [M⁺].

Example 118{(4-Methyl-5-(5-methylsulfamoyl-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carbonyl]-amino)-aceticacid

A mixture of [(5-formyl-4-methyl-1H-pyrrole-3-carbonyl)-amino]-aceticacid ethyl ester (0.142 g, 0.59 mmol) and 1N NaOH (1.2 mL) in methanol(10 mL) was stirred at room temperature for 1 hr. The reaction wasconcentrated and the residue was condensed with5-methylaminosulfonyl-2-oxindole (0.13 g, 0.48 mmol) and piperidine(0.12 mL) in ethanol (12 mL) to give 0.11 g (52%) of the title compound.

¹HNMR (300 MHz, DMSO-d6) δ 13.98 (br s, 1H, NH), 8.17 (s, 1H), 7.80 (s,1H), 7.75 (d, J=3.1 Hz, 1H), 7.51 (dd, J=2 & 8.2 Hz, 1H), 7.21 (m on brs, 2H), 6.97 (d, J=8.1 Hz, 1H), 3.41 (d, J=4.2 Hz, 2H, CH₂NH), 2.54 (s,3H, pyrrole-CH₃), 2.39 (s, 3H, ArCH ₃).

MS m/z 417 [M−1]⁺.

Example 1205-Methyl-2-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid

¹HNMR (300 MHz, DMSO-d6) δ 13.77 (br s, 1H, NH), 12.49 (s, 1H, COOH),11.07 (s, 1H, NH), 8.39 (s, 1H, H-vinyl), 7.43 (d, J=7.47 Hz, 1H), 7.20(t, J=7.47 Hz, 1H), 7.03 (t, J=7.47 Hz, 1H), 6.91 (d, J=7.47 Hz, 1H),6.49 (d, J=1.53 Hz, 1H), 2.34 (s, 3H, CH₃).

MS m/z 269 [M+H]⁺.

Example 1215-Methyl-2-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid ethyl ester

¹HNMR (300 MHz, DMSO-d6) δ 13.79 (s, 1H, NH), 11.08 (s, 1H, NH), 8.31(s, 1H, H-vinyl), 7.45 (d, J=7.52 Hz, 1H), 7.20 (t, J=7.52 Hz, 1H), 7.03(t, J,=7.52 Hz, 1H), 6.91 (d, J=7.52 Hz, 1H), 6.50 (d, J=2.1 Hz, 1H),4.26 (q, J=7.2 Hz, 2H, OCH₂CH₃), 2.33 (s, 3H, CH₃), 1.32 (t, J=7.2 Hz,3H, OCH₂CH₃).

MS m/z 297.1 [M+H]⁺.

Example 1222-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-5-methyl-1H-pyrrole-3-carboxylicacid ethyl ester

¹HNMR (360 MHz, DMSO-d6) δ 13.72 (s, 1H, NH), 11.16 (s, 1H, NH), 8.29(s, 1H, H-vinyl), 7.53 (d, J=2.0 Hz, 1H), 7.35 (dd, J=2.0 & 8.05 Hz,1H), 6.87 (t, J=8.05 Hz, 1H), 6.53 (d, J=2.4 Hz, 1H), 4.28 (q, J=7.03Hz, 2H, OCH₂CH₃), 2.35 (s, 3H, CH₃), 1.33 (t, J=7.03 Hz, 3H, OCH₂CH₃).

MS m/z 375 & 377 [M+H]⁺.

Example 1232-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-5-methyl-1H-pyrrole-3-carboxylicacid

¹HNMR (300 MHz, DMSO-d6) δ 13.72(s, 1H, NH), 12.57 (s, 1H, COOH), 11.19(s, 1H, NH), 8.36 (s, 1H, H-vinyl), 7.51 (d, J=1.4 Hz, 1H), 7.34 (dd,J=1.4 & 8.17 Hz, 1H), 6.87 (t, J=8.17 Hz, 1H), 6.52 (d, J=2.5 Hz, 1H),2.35 (s, 3H, CH₃).

MS m/z 347 & 349 [M+H]⁺.

Example 1242-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-5-methyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ylethyl)-amide

To a solution of 2-formyl-5-methyl-1H-pyrrole-3-carboxylic acid (250 mg,1.63 mmol) in dimethylformamide (3 mL) was added1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (376 mg, 1.2 equiv.),1-hydroxybenzotriazole (265 mg, 1.2 equiv.), triethylamine (0.45 mL, 2equiv.) and 1-(2-aminoethyl)pyrrolidine (0.23 mL. 1.1 equiv.). Afterstirring at room temperature overnight, the reaction was diluted withsaturated sodium bicarbonate and brine (with extra salt) and extractedwith 10% methanol in dichloromethane. The combined organic layers werewashed with brine, dried over anhydrous sodium sulfate and concentratedto give 130 mg of 2-formyl-5-methyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-yl-ethyl)-amide.

A mixture of 5-bromo-2-oxindole (106 mg, 0.5 mmol),2-formyl-5-methyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-yl-ethyl)-amide (125 mg, 1 equiv.) and piperidine (0.2mL) in ethanol (2 mL) was heated in a sealed tube at 80° C. for 1 hr andthen cooled. The precipitate which formed was collected by vacuumfiltration, washed with ethanol and ethyl acetate and dried to give thetitle compound as an orange solid.

¹HNMR (300 MHz, DMSO-d6) δ 13.62 (s, 1H, NH), 11.06 (br s, 1H, NH), 8.56(s, 1H, H-vinyl), 8.15 (m, 1H, CONHCH₂), 7.48 (d, J=1.8 Hz, 1H), 7.31(dd, J=1.8 & 7.9 Hz, 1H), 6.86 (d, J=7.9 Hz, 1H), 6.60 (d, J=2.3 Hz,1H), 3.35 (m, 2H, HNCH₂CH₂), 2.56 (t, J=6.91 Hz, 2H, HNCH₂CH₂), 2.35 (s,3H, CH₃), 1.67 (m, 4H, 2×CH₂).

MS m/z 443/445 [M⁺ and M⁺+2].

Example 1252-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-5-methyl-1H-pyrrole-3-carboxylicacid (2-diethylaminoethyl)-amide

To a solution of 2-formyl-5-methyl-1H-pyrrole-3-carboxylic acid (320 mg,2.1 mmol) in dimethylformamide (3 mL) was added1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (483 mg, 1.2 equiv.),1-hydroxybenzotriazole (340 mg, 1.2 equiv.), triethylamine (0.59 mL, 2equiv.) and N,N-diethylethylenediamine (0.32 mL, 1.1 equiv.). Afterstirring at room temperature overnight, the reaction was diluted withsaturated sodium bicarbonate and brine (with extra salt) and extractedwith 10% methanol in dichloromethane. The combined organic layers werewashed with brine, dried over anhydrous sodium sulfate and concentratedto give 2-formyl-5-methyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)-amide.

A mixture of 5-bromo-2-oxindole (106 mg, 0.5 mmol),2-formyl-5-methyl-1H-pyrrole-3-carboxylic acid(2-diethylamino-ethyl)-amide (126 mg, 1 equiv.) and piperidine (0.2 mL)in ethanol (2 mL) was heated in a sealed tube at 80° C. for 1 hr andthen cooled. The precipitate was collected by vacuum filtration, washedwith ethanol and ethyl acetate and dried to give the title compound asan orange solid.

¹HNMR (360 MHz, DMSO-d6) δ 13.62 (s, 1H, NH), 11.11 (br s, 1H, NH), 8.54(s, 1H, H-vinyl), 8.1 (m, 1H, CONHCH₂), 7.49 (d, J=2.2 Hz, 1H), 7.31(dd, J=2.2 & 8.3 Hz, 1H), 6.86 (d, J=8.3 Hz, 1H), 6.58 (d, J=2.24 Hz,1H), 3.31 (m, 2H, HNCH₂CH₂), 2.59 (m, 6H, 3×CH₂), 2.36 (s, 3H, CH₃),0.99 (t, J=6.8 Hz, 6H, N(CH₂CH₃)₂).

MS m/z 445/447 [M⁺ and M⁺+2].

Example 1262,4-Dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)-amide

A mixture of 1,3-dihydro-indol-2-one (266 mg, 2 mmol),5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylamino-ethyl)-amide (530 mg, 2 mmol) and piperidine (1 drop) inethanol was heated at 90° C. for 2 hours. The reaction was cooled toroom temperature, the resulting precipitate was collected by vacuumfiltration, washed with ethanol and dried to give 422 mg (55%) of thetitle compound as a light yellow solid.

¹H NMR (400 MHz, DMSO-d6) δ 13.7 (s, 1H, NH), 10.9 (s, 1H, NH), 7.88 (d,J=7.6 Hz, 1H), 7.64 (s, 1H, H-vinyl), 7.41 (t, J=5.4 Hz, 1H, NH), 7.13(dt, J=1.2 & 7.6 Hz, 1H), 6.99 (dt, J=1.2 & 7.6 Hz, 1H), 6.88 (d, J=7.6Hz, 1H), 3.28 (m, 2H), 2.48-2.55 (m, 6H), 2.44 (s, 3H, CH₃), 2.41 (s,3H, CH₃), 0.97 (t, J=7.2 Hz, 6H, N(CH₂CH₃)₂).

MS+ve APCI 381 [M⁺+1].

Example 1275-(5-Chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)-amide

A mixture of 5-Chloro-1,3-dihydro-indol-2-one (335 mg, 2 mmol),5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylamino-ethyl)-amide (530 mg, 2 mmol) and piperidine (1 drop) inethanol was heated at 90° C. for 2 hours. The reaction was cooled toroom temperature; the resulting precipitate was collected by vacuumfiltration, washed with ethanol and dried to give 565 mg (68%) of thetitle compound as an orange solid.

¹H NMR (400 MHz, DMSO-d6) δ 13.65 (s, 1H, NH), 11.0 (s, 1H, NH), 7.98(d, J=2.1 Hz, 1H) 7.77 (s, 1H H-vinyl), 7.44 (t, NH), 7.13 (dd, J=2.1 &8.4 Hz, 1H) 6.87 (d, J=8.4 Hz, 1H), 3.28 (g, 2H), 2.48-2.53 (m, 6H),2.44 (s, 3H, CH₃), 2.43 (s, 3H, CH₃), 0.97 (t, J=7.0 Hz, 6H, N(CH₂CH₃)₂)

MS+ve APCI 415 [M⁺+1].

Example 1282,4-Dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-ethyl)-amide

1,3-Dihydro-indol-2-one was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-yl-ethyl)-amide to give the title compound.

MS+ve APCI 379 [M⁺+1].

Example 1295-(5-Fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-yl-ethyl)-amide

5-Fluoro-1,3-dihydro-indol-2-one was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-yl-ethyl)-amide to give the title compound.

MS+ve APCI. 397 [M⁺+1].

Scale-Up Procedure:

5-Formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (61 g),5-fluoro-1,3-dihydro-indol-2-one (79 g), ethanol (300 mL) andpyrrolidine (32 mL) were refluxed for 4.5 hours. Acetic acid (24 mL) wasadded to the mixture and refluxing was continued for 30 minutes. Themixture was cooled to room temperature and the solids collected byvacuum filtration and washed twice with ethanol. The solids were stirredfor 130 minutes in 40% acetone in water (400 mL) containing 12 Nhydrochloric acid (6.5 mL). The solids were collected by vacuumfiltration and washed twice with 40% acetone in water. The solids weredried under vacuum to give5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (86 g, 79% yield) as an orange solid. ¹H-NMR (dimethylsulfoxide-d₆)δ 2.48, 2.50 (2×s, 6H, 2×CH₃), 6.80, 6.88, 7.68, 7.72 (4×m, 4H, aromaticand vinyl), 10.88 (s, 1H, CONH), 12.12 (s, 1H, COOH), 13.82 (s, 1H,pyrrole NH). MS m/z 299 [M−1].

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (100 g) and dimethylformamide (500 mL) were stirred andbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(221 g), 1-(2-aminoethyl)pyrrolidine (45.6 g) and triethylamine (93 mL)were added. The mixture was stirred for 2 hours at ambient temperature.The solid product was collected by vacuum filtration and washed withethanol. The solids were slurry-washed by stirring in ethanol (500 mL)for one hour at 64° C. and cooled to room temperature. The solids werecollected by vacuum filtration, washed with ethanol, and dried undervacuum to give5-(5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-yl-ethyl)-amide (101.5 g, 77% yield). ¹H-NMR(dimethylsulfoxide-d₆) δ 1.60 (m, 4H, 2×CH₂), 2.40, 2.44 (2×s, 6H,2×CH₃), 2.50 (m, 4H, 2×CH₂), 2.57, 3.35 (2×m, 4H, 2×CH₂), 7.53, 7.70,7.73, 7.76 (4×m, 4H, aromatic and vinyl), 10.88 (s, 1H, CONH), 13.67 (s,1H, pyrrole NH). MS m/z 396 [M+1].

Example 1305-(5-Chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-yl-ethyl)-amide

5-Chloro-1,3-dihydro-indol-2-one was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-yl-ethyl)-amide to give the title compound.

MS+ve APCI 413 [M⁺+1].

Example 1312,4-Dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid (2-dimethylaminoethyl)-amide

1,3-Dihydro-indol-2-one was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-dimethylamino-ethyl)amide to give the title compound.

¹H NMR (400 MHz, PMSO-d6) δ 13.63 (s, 1H, NH), 10.90 (s, 1H, NH), 7.78(d, J=7.8 Hz, 1H), 7.63 (s, 1H H-vinyl), 7.48 (t, 1H, NH), 7.13 (dt,1H), 6.98 (dt, 1H), 6.88 (d, J=7.7 Hz, 1H), 3.31 (q, J=6.6 Hz, 2H), 2.43(s, 3H, CH₃), 2.40 (s, 3H, CH₃), 2.38 (t, J=6.6 Hz, 2H), 2.19 (s, 6H,N(CH₂CH₃)₂)

MS+ve APCI 353 [M⁺+1].

Example 1325-(5-Fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-dimethylaminoethyl)-amide

5-Fluoro-1,3-dihydro-indol-2-one was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-dimethylaminoethyl)amide to give the title compound.

¹H NMR (400 MHz, DMSO-d6) δ 13.68 (s, 1H, NH), 10.90 (s, 1H, NH), 7.76(dd, J=2.4 & 9.4 Hz, 1H), 7.71 (s, 1H H-vinyl), 7.51 (t, 1H, NH), 6.93(m, 1H), 6.84 (dd, J=4.6 & 8.4 Hz, 1H), 3.31 (q, J=6.6 Hz, 2H), 2.43 (s,3H, CH₃), 2.41 (s, 3H, CH₃), 2.38 (t, J=6.6 Hz, 2H), 2.19 (s, 6H,N(CH₂CH₃)₂)

MS+ve APCI 371 [M⁺+1].

Example 1935-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-ethylamino-ethyl)-amide

5-Formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-ethylamino-ethyl)-amide (99 g), ethanol (400 mL), 5-fluoro-2-oxindole(32 g) and pyrrolidine (1.5 g) were refluxed for 3 hours with stirring.The mixture was cooled to room temperature and the solids collected byvacuum filtration. The solids were stirred in ethanol at 60° C., cooledto room temperature and collected by vacuum filtration. The product wasdried under vacuum to give5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-ethylamino-ethyl)-amide (75 g, 95% yield). ¹H-NMR(dimethylsulfoxide-d₆) δ 1.03 (t, 3H, CH₃), 2.42, 2.44 (2×s, 6H, 2×CH₃),2.56 (q, 2H, CH₂), 2.70, 3.30 (2×t, 4H, 2×CH₂), 6.85, 6.92, 7.58, 7.72,7.76 (5×m, 5H, aromatic, vinyl and CONH), 10.90 (br s, 1H, CONH), 13.65(br s, 1H, pyrrole NH).

MS m/z 369 [M−1].

Example 1955-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethyl-N-oxoamino-ethyl)-amide

Method A:

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)-amide (598 mg) and dichloromethane (60 mL)in an ice bath were treated with 3-chloroperbenzoic acid (336 mg) andthe mixture stirred at room temperature overnight. The solvent wasrotary evaporated and the residue suspended in methanol (20 mL). Water(20 mL) containing sodium hydroxide (240 mg) was added and the mixturestirred for one hour. The precipitate was collected by vacuumfiltration, washed with 5 mL of water and dried under a vacuum to give5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethyl-N-oxoamino-ethyl)-amide (510 mg, 82% yield) as an orangesolid. ¹H-NMR (DMSO-d6) δ 13.72 (br s, 1H, NH), 11.02 (br s, 1H, CONH),9.81 (br s, 1H, CONH), 7.75 (dd, 1H, aromatic), 7.70 (s, 1H, aromatic),6.93 (td, 1H, aromatic), 6.84 (m, 1H, aromatic), 3.63 (m, 2H, CH₂), 3.29(m, 2H, CH₂), 3.14 (m, 4H, 2×CH₂), 2.47 (s, 1H, CH₃), 2.45 (s, 3H, CH₃),1.64 (t, 6H, 2×CH₃). MS m/z 415 [M+1].

Method B:

5-Formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylamino-ethyl)-amide (10 g) was suspended in dichloromethane(100 mL) and cooled in an ice bath. 3-Chloro-peroxybenzoic acid (13.1 g)was added with stirring and the mixture allowed to warm to roomtemperature and then stirred ovenight. The mixture was rotary evaporatedto dryness and chromatographed on a column of silica gel eluting with20% methanol in dichloromethane. Fractions containing product werecombined and rotary evaporated to dryness to give5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethyl-N-oxoamino-ethyl)-amide (9 g, 83% yield).

5-Formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethyl-N-oxoamino-ethyl)-amide (9 g),5-fluoro-1,3-dihydro-indol-2-one ((9 g, 83% yield)), and pyrrolidine ((9g, 83% yield (0.1 g) were refluxed in ethanol (30 mL) for 4 hours. Themixture was cooled in an ice bath and the precipitate collected byvacuum filtration and washed with ethanol. The solids were stirred inethyl acetate (30 mL), collected by vacuum filtration, washed with ethylacetate and dried under vacuum to give5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethyl-N-oxoamino-ethyl)-amide (10.3 g 80% yield) as an orangesolid. ¹H-NMR (DMSO-d6) δ 13.72 (br s, 1H, NH), 11.02 (br s, 1H, CONH),9.81 (br s, 1H, CONH), 7.75 (dd, 1H, aromatic), 7.70 (s, 1H, aromatic),6.93 (td, 1H, aromatic), 6.84 (m, 1H, aromatic), 3.63 (m, 2H, CH₂),3.29. (m, 2H, CH₂), 3.14 (m, 4H, 2×CH₂), 2.47 (s, 1H, CH₃), 2.45 (s, 3H,CH₃), 1.64 (t, 6H, 2×CH₃). MS m/z 415 [M+1].

Example 1905-(5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-(pyridin-1-yl)ethyl]-amide.

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (120 mg, 0.4 mmol) was shaken with EDC, HCl (96 mg, 0.5 mmol),anhydrous 1-hydroxy-benztriazole (68 mg, 0.5 mmol), and2-(2-aminoethylpyridine purchased from Aldrich in anhydrous DMF (3 mL)for 2-3 days at room temperature. The reaction mixture was diluted with1M NaHCO3 (1.5 ml), then with 8 ml of water. The precipitated crudeproduct was collected by filtration, washed with water, dried andpurified by crystallization or chromatography to give5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid [2-(pyridin-1-yl)-ethyl]amide.

Example 1895-[5-Chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-(pyridin-1-yl)ethyl]amide

Proceeding as described in previous example but substituting5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid with5-[5-chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (127 mg)-provided5-(5-chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid [2-(pyridin-1-yl)ethyl]amide.

Example 1925-[5-Bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid [2-(pyridin-1-yl)ethyl]amide

Proceeding as described in Example 190 above but substituting5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid with5-(5-bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (145 mg) provided5-(5-bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid [2-(pyridin-1-yl)ethyl]amide.

Example 1915-[2-Oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid [2-(pyridin-1-yl)ethyl]amide

Proceeding as described in Example 190 above but substituting5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidene-methyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid with5-(2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (113 mg) provided5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid [2-(pyridin-1-yl)ethyl]amide.

Example 2035-[5-Cyano-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid [2-(pyridin-1-yl)ethyl]amide

Proceeding as described in Example 190 above but substituting5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid with5-[5-cyano-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (123 mg) provided5-[5-cyano-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-1-dimethyl-1H-pyrrole-3-carboxylicacid [2-(pyridin-1-yl)ethyl]amide.

Examples 142, 186, 187, 188 and 204

Proceeding as described in Examples 190, 189, 191, 192, and 203 abovebut substituting 2-(2-aminoethyl)pyridine with1-(2-aminoethyl)pyrrolidine, purchased from Aldrich Chemical Company,Inc. provided the desired compounds.

Examples 143-147

Proceeding as described in Examples 190, 189, 191, 192, and 203 abovebut substituting 2-(2-aminoethyl)pyridine with1-(2-aminoethyl)imidazolin-2-one (prepared by heating dimethyl carbonatewith bis(2-aminoethyl) amine (2 equivalents) in a sealed flask to 150°C. for 30 min., following the procedure described in U.S. Pat. No.2,613,212 (1950), to Rohm & Haas Co. The crude product was purified onsilica using an eluent mixture chloroform-methanol-aqueous ammonia80:25:2) provided the desired compounds.

Examples 148-151 and 184

Proceeding as described in Examples 190, 189, 191, 192, and 203 abovebut substituting 2-(2-aminoethyl)pyridine with4-(2-aminoethyl)piperazine-1-acetic acid ethyl ester (prepared asfollows: Piperazine-1-acetic acid ethyl ester (11.22 g) was treated withiodoacetonitrile (5.0 mL) in the presence of potassium carbonate (6.9 g)in ethyl acetate (260 mL) at 0° C. After complete iodoacetonitrileaddition (45 min), the reaction mixture was subsequently stirred at roomtemperature for 11 hours. The reaction mixture was filtered and thefiltrates evaporated. The residue was hydrogenated in a presence ofcobalt boride (prepared from CoCl2 and sodium borohydride) at roomtemperature at 50 psi for 2 days in ethanol. Filtration, evaporation andchromatographic purification using an eluent mixturechloroform-methanol-aqueous ammonia 80:25:2 provided the desired amine(3.306 g) as a pale yellow oil) provided the desired compounds.

Example 152-153

Proceeding as described in Examples 190, 189, 191, 192, and 203 abovebut substituting 2-(2-aminoethyl)pyridine with2-[(2-aminoethylamino)]acetonitrile (prepared as follows: A solution ofiodoacetonitrile (50 mmol) in ethyl alcohol (80 ml) was added to asolution of ethylene diamine (150 ml) in ethyl alcohol (60 ml) at 0° C.over a period of 30 minutes. The stirring was continued for another 1 hrat 0° C., then at room temperature for 14 hours. 55 mmol of potassiumcarbonate was added, stirred for 30 minutes, filtered and the filtratewas concentrated at room temperature. The residue, was purified onsilica using an eluent mixture chloroform-methanol-aqueous ammonia80:15:1.5 to give 2-[(2-aminoethylamino)]-acetonitrile (3.550 g) whichwas used immediately) provided the desired compounds.

Example 154-158

Proceeding as described in Examples 190, 189, 191, 192, and 203 abovebut substituting 2-(2-aminoethyl)pyridine with1-(3-aminopropyl)-azepin-2-one (prepared according to the procedure inKraft A.: J. Chem. Soc. Perkin Trans. 1, 6, 1999, 705-14, except thatthe hydrolysis of DBU was performed at 145° C. neat in a presence oflithium hydroxide (1 hr, 5 ml of DBU, 2 ml of water, 420 mg of lithiumhydroxyde hydrate). Purification of the crude product on silica using aneluent mixture chloroform-methanol-aqueous ammonia 80:40:4 provided1-(3-aminopropyl)azepin-2-one (4.973 g, 87% yield)) provide the desiredcompounds.

Examples 133-135, 159 and 200

Proceeding as described in Examples 190, 189, 191, 192, and 203 abovebut substituting 2-(2-aminoethyl)pyridine with N-acetyl ethylenediamine, (prepared by heating a mixture of ethyl acetate with ethylenediamine (1.5 equivalents) to 160° C. for 1 hr in a sealed vessel. Thevacuum distillation provided the desired product in 56% yield.N-acetylethylene diamine is also available from Aldrich) provide thedesired compounds.

Examples 146-140

Proceeding as described in Examples 190, 189, 191, 192, and 203 abovebut substituting 2-(2-aminoethyl)pyridine with1-(3-aminopropyl)-tetrahydro-pyrimidin-2-one (prepared in the same wayas 1-(3-aminopropyl)-azepin-2-one according to the procedure in KraftA.: J. Chem. Soc. Perkin Trans. 1, 6, 1999, 705-14: Briefly,1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (4.939 g), lithiumhydroxyde hydrate (918 mg) and 2 ml of water was heated without asolvent in a sealed vessel to 145° C. for 1 hr. The crude product waspurified on a column of silica in chloroform-methanol-aqueous ammonia80:40:4 to give pure amine (5.265 g, 94% yield).

Examples 141, 160-162 and 185

Proceeding as described in Examples 190, 189, 191, 192, and 203 abovebut substituting 2-(2-aminoethyl)pyridine with1-(2-aminoethyl)-piperazine-2-one (prepared as follows: Neattert-butyldiphenylsilyl chloride (25 mL, 97.7 mmol) was added dropwiseinto a solution of DBU (19.5 ml, 130 mmol) and bis(2-aminoethyl)amine(4.32 mL, 40 mmol) in anhydrous dimethyl acetamide (80 mL) at roomtemperature upon cooling on water bath within 5 minutes. The mixture wasstirred for 5 hours. Bromoacetic acid ethyl ester (6.70 mL, 60 mmol) wasadded neat upon cooling to room temperature. The reaction was stirredfor 25 minutes, then evaporated on high vacuum. The residue wasdissolved in methanol (200 ml), KHCO₃ (10 g) and KF (12 g, 200 mmol)were added and the mixture was stirred at 60° C. for 5 hours. 10 g ofNa₂CO₃ was added, stirred for 10 minutes, cooled and filtered. Thefiltrates were evaporated. The residue was extracted with hexanes (2times 250 ml). The hexane-insoluble material was dissolved in ethanol(60 ml), filtered and evaporated. The residue was purified on a columnof silica in chloroform-methanol-aqueous ammonia 80:40:4 to give pureamine (4.245 g, 74% yield)) provided the desired compounds.

Examples 163-167

Proceeding as described in Examples 190, 189, 191, 192, and 203 abovebut substituting 2-(2-aminoethyl)pyridine with3-[(2-aminoethyl)amino]propionitrile (prepared from ethylene diamine(150 mmol) and acrylonitrile (50 mmol) in THF at room temperature, asdescribed in Israel, M. et al: J. Med Chem. 7, 1964, 710-16., providedthe desired amine (4.294 g)) provided the desired compounds.

Example 1685-(5-Fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-(4-methylpiperazin-1-yl)-ethyl]-amide

To a stirred yellow muddy mixture of5-(5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (90 mg), DMF (0.8 mL) and TEA (0.084 mL) in a 20 mL reaction tube,was added BOP reagent (199 mg). The mixture became clear in 5 min.2-(4-Methylpiperazin-1-yl)ethylamine¹ (51 mg) was added into the clearmixture. The resulting solution was stirred at room temperature overnight. Yellow solid products precipitated from the reaction system. Thinlayer chromatography (10% methanol in methylene chloride) showed thatall the starting material had been converted into the product. The solidwas isolated by vacuum filtration and washed once with ethanol (1 mL).The solid was sonicated in diethyl ether (2 mL) for 20 min and collectedby vacuum filtration. After drying under vacuum,5-(5-Fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (4-methylpiperazin-1-yl-ethyl)-amide (79 mg, 62% yield) wasobtained.

¹H NMR (DMSO-d₆) δ 2.13 (s, 3H, CH₃), 2.40, 2.42 (2×s, 6H, 2×CH₃), 2.41(m, 2H, CH₂), 2.47 (m, 8H, 4×CH₂), 3.30 (m, 2H, CH₂), 6.82 (dd, J=4.5,8.7 Hz, 1H), 6.91 (td, ²J=2.4; ³J=8.8 Hz, 1H), 7.43 (t, J=5.6 Hz, 1H),7.70 (s, 1H), 7.75 (dd, J=2.8, 9.6 Hz, 1H) (aromatic and vinyl), 10.88(s, 1H, CONH), 13.67 (s, 1H, NH). LC-MS (m/z) 424.4 (M−1).

Example 1695-(5-Chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (4-methylpiperazin-1-yl-ethyl)-amide

Following the procedure in Example 168 above but substituting5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid with5-[5-chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (95 mg, 0.3 mmol) gave5-(5-chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (4-methylpiperazin-1-yl-ethyl)-amide (76 mg, 58%).

¹H NMR (DMSO-d₆) δ 2.13 (s, 3H, CH₃), 2.41, 2.42 (2×s, 6H, 2×CH₃), 2.42(m, 2H, CH₂), 2.48 (m, 8H, 4×CH₂), 3.30 (m, 2H, CH₂), 6.84 (d, J=8.0 Hz,1H), 7.11 (dd, J=2.0, 8.0 Hz, 1H), 7.44 (t, J=5.6 Hz, 1H), 7.76 (s, 1H),7.97 (d, J=2.0 Hz, 1H) (aromatic and vinyl), 10.98 (s, 1H, CONH), 13.62(s, 1H, NH). LC-MS (m/z) 440.2 (M−1).

Example 1705-(5-Bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (4-methylpiperazin-1-yl-ethyl)-amide

Following the procedure described in Example 168, but substituting5-(5-chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid with5-(5-bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid gave5-(5-bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (4-methylpiperazin-1-yl-ethyl)-amide (39 mg, 54%). was obtainedfrom SU011670 (54 mg, 0.15 mmol).

¹H NMR (DMSO-d₆) δ 2.14 (s, 3H, CH₃), 2.41, 2.42 (2×s, 6H, 2×CH₃), 2.42(m, 2H, CH₂), 2.48 (m, 8H, 4×CH₂), 3.31 (m, 2H, CH₂), 6.80 (d, J=8.0 Hz,1H), 7.23 (dd, J=2.0, 8.0 Hz, 1H), 7.44 (t, J=5.6 Hz, 1H), 7.76 (s, 1H),8.09 (d, J=2.0 Hz, 1H) (aromatic and vinyl), 10.99 (s, 1H, CONH), 13.61(s, 1H, NH). LC-MS (m/z) 486.6 (M).

Example 1725-(2-Oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (4-methylpiperazin-1-yl-ethyl)-amide

Following the procedure described in Example. 168 above but substituting5-(5-fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid SU014900 with5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid gave5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (4-methylpiperazin-1-yl-ethyl)-amide, SU014903 (136 mg, 84%) wasobtained from SU012120 (112.8 mg, 0.4 mmol)

¹H-NMR (DMSO-d₆) δ 2.13 (s, 3H, CH₃), 2.39, 2.42 (2×s, 6H, 2×CH₃), 2.42(m, 2H, CH₂), 2.48 (m, 8H, 4×CH₂), 3.30 (t, 2H, CH₂), 6.86 (d, J=8.0 Hz,1H), 6.96 (t, J=7.4 Hz, 1H), 7.10 (t, J=7.8 Hz, 1H), 7.41 (t, J=5.4 Hz,1H), 7.62 (s, 1H), 7.76 (d, J=7.6 Hz, 1H) (aromatic and vinyl), 10.88(s, 1H, CONH), 13.61 (s, 1H, NH). LC-MS (m/z) 406.6 (M−1).

Example 1715-(2-Oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-(3,5-dimethylpiperazin-1-yl)ethyl)amide

To a stirred yellow muddy mixture of5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (112.8 mg, 0.4 mmol), DMF (0.5 mL) and triethylamine (0.111 mL) ina 20 mL reaction tube, was added BOP reagent (265 mg). The mixturebecame clear in 5 min. 2-(2,6-dimethylpiperazin-1-yl)ethylamine (68.6mg) (see., Tapia, L. Alonso-Cires, P. Lopez-Tudanca, R. Mosquera, L.Labeaga, A. Innerarity, A. Orjales, J. Med. Chem., 1999, 42, 2870-2880)was added into the clear mixture. The resulting solution was stirred atroom temperature over night. Thin layer chromatography (10% methanol inmethylene chloride) showed that all the starting material had beenconverted into the product. The reaction mixture was evaporated todryness and then purified by flash chromatography(CH₂Cl₂/CH₃OH=20/1-15/1) followed by recrystalization to give5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid [2-(3,5-dimethylpiperazin-1-yl)ethyl)amide (83 mg, 50% yield).

¹H NMR (DMSO-d₆) δ 1.15, 1.16 (2×s, 6H, 2×CH₃), 1.95 (t, J=11.6 Hz, 2H,CH₂), 2.41, 2.47 (2×s, 6H, 2×CH₃), 2.50 (m, 2H, CH₂), 3.03 (d, J=10 Hz,2H), 3.19 (m, 2H), 3.30 (m, 2H, CH₂), 6.86 (d, J=8.0 Hz, 1H), 6.97 (t,J=7.2 Hz, 1H), 7.11 (t, J=7.8 Hz, 1H), 7.48 (t, J=5.6 Hz, 1H), 7.61 (s,1H), 7.75 (d, J=7.6 Hz, 1H) (aromatic and vinyl), 10.88 (s, 1H, CONH),13.62 (s, 1H, NH). LC-MS (m/z) 422.2 (M+1).

Example 173 5-(5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-(3,5-dimethylpiperazin-1-yl)ethyl)amide

Following the procedure described in Example 168 above the desiredcompound was obtained (60 mg, 0.2 mmol).

¹H NMR (DMSO-d₆) δ 0.891, 0.907 (2×s, 6H, 2×CH₃), 1.49 (t, J=10.4 Hz,2H), 2.40, 2.42 (2×s, 6H, 2×CH₃), 2.41 (m, 2H, CH₂), 2.74 (m, 4H), 3.30(m, 2H), 6.82 (dd, J=4.5, 8.7 Hz, 1H), 6.90 (td, ²J=2.4, ³J=8.4 Hz, 1H),7.42 (t, J=5.6 Hz, 1H), 7.70 (s, 1H), 7.74 (dd, J=4.6, 8.4 Hz, 1H)(aromatic and vinyl), 10.88 (s, 1H, CONH), 13.65 (s, 1H, NH). LC-MS(m/z) 438.4 (M−1).

Example 1745-[5-Chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid [2-(3,5-dimethylpiperazin-1-yl)ethyl)amide

Following the procedure for Example 171 above the desired compound (31.2mg, 34%) was obtained from5-[5-chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (63 mg, 0.2 mmol).

¹H NMR (DMSO-d₆) δ 1.15, 1.16 (2×s, 6H, 2×CH₃), 1.95 (t, J=11.6 Hz, 2H,CH₂), 2.40, 2.42 (2×s, 6H, 2×CH₃), 2.50 (m, 2H, CH₂), 3.03 (d, J=11.2Hz, 2H), 3.19 (m, 2H), 3.30 (m, 2H, CH₂), 6.85 (d, J=8.4 Hz, 1H), 7.11(dd, J=2.0, 8.0 Hz,1H), 7.52 (t, J=5.6 Hz, 1H), 7.76 (s, 1H), 7.97 (d,J=2.0 Hz, 1H) (aromatic and vinyl), 10.99 (s, 1H, CONH), 13.63 (s, 1H,NH). LC-MS (m/z) 456.2 (M+1).

Example 1755-[5-Bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid [2-(3,5-dimethylpiperazin-1-yl)ethyl)amide

Following the procedure described in Example 171 the desired compound(40 mg, 40%) was obtained from5-[5-bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (74 mg, 0.2 mmol).

¹H NMR (DMSO-d₆) δ 1.15, 1.16 (2×s, 6H, 2×CH₃), 1.95 (t, J=11.6 Hz, 2H,CH₂), 2.40, 2.42 (2×s, 6H, 2×CH₃), 2.50 (m, 2H, CH₂), 3.03 (d, J=10.4Hz, 2H), 3.19 (m, 2H), 3.30 (m, 2H, CH₂), 6.81 (d, J=8.4 Hz, 1H), 7.24(dd, J=2.0, 8.4 Hz, 1H), 7.51 (t, J=5.6 Hz, 1H), 7.76 (s, 1H), 8.10 (d,J=2.0 Hz, 1H) (aromatic and vinyl), 10.99 (s, 1H, CONH), 13.62 (s, 1H,NH). LC-MS (m/z) 498.4 (M−1).

Biological Examples

The following assays are employed to find those compounds demonstratingthe optimal degree of the desired activity.

A. Assay Procedures.

The following assays may be used to determine the level of activity andeffect of the different compounds of the present invention on one ormore of the PKs. Similar assays can be designed along the same lines forany PK using techniques well known in the art.

Several of the assays described herein are performed in an ELISA(Enzyme-Linked Immunosorbent Sandwich Assay) format (Voller, et al.,1980, “Enzyme-Linked Immunosorbent Assay,” Manual of ClinicalImmunology, 2d ed., Rose and Friedman, Am. Soc. Of Microbiology,Washington, D.C., pp. 359-371). The general procedure is as follows: acompound is introduced to cells expressing the test kinase, eithernaturally or recombinantly, for a selected period of time after which,if the test kinase is a receptor, a ligand known to activate thereceptor is added. The cells are lysed and the lysate is transferred tothe wells of an ELISA plate previously coated with a specific antibodyrecognizing the substrate of the enzymatic phosphorylation reaction.Non-substrate components of the cell lysate are washed away and theamount of phosphorylation on the substrate is detected with an antibodyspecifically recognizing phosphotyrosine compared with control cellsthat were not contacted with a test compound.

The presently preferred protocols for conducting the ELISA experimentsfor specific PKs is provided below. However, adaptation of theseprotocols for determining the activity of compounds against other RTKS,as well as for CTKs and STKs, is well within the scope of knowledge ofthose skilled in the art. Other assays described herein measure theamount of DNA made in response to activation of a test kinase, which isa general measure of a proliferative response. The general procedure forthis assay is as follows: a compound is introduced to cells expressingthe test kinase, either naturally or recombinantly, for a selectedperiod of time after which, if the test kinase is a receptor, a ligandknown to activate the receptor is added. After incubation at leastovernight, a DNA labeling reagent such as 5-bromodeoxyuridine (BrdU) orH³-thymidine is added. The amount of labeled DNA is detected with eitheran anti-BrdU antibody or by measuring radioactivity and is compared tocontrol cells not contacted with a test compound.

GST-FLK-1 Bioassay

This assay analyzes the tyrosine kinase activity of GST-Flk1 onpoly(glu,tyr) peptides.

Materials and Reagents:

1. Corning 96-well ELISA plates (Corning Catalog No. 5805-96).

2. poly(glu,tyr) 4:1, lyophilizate (Sigma Catalog #P0275).

3. Preparation of poly(glu,tyr)(pEY) coated assay plates: Coat 2 ug/wellof poly(glu,tyr)(pEY) in 100 ul PBS, hold at room temperature for 2hours or at 4° C. overnight. Cover plates well to prevent evaporation.

4. PBS Buffer: for 1 L, mix 0.2 g KH₂PO₄, 1.15 g Na₂HPO₄, 0.2 g KCl and8 g NaCl in approx. 900 ml dH₂O. When all reagents have dissolved,adjust the pH to 7.2 with HCl. Bring total volume to 1 L with dH₂O.

5. PBST Buffer: to 1 L of PBS Buffer, add 1.0 ml Tween-20.

6. TBB—Blocking Buffer: for 1 L, mix 1.21 g TRIS, 8.77 g NaCl, 1 mlTWEEN-20 in approximately 900 ml dH₂O. Adjust pH to 7.2 with HCl. Add 10g BSA, stir to dissolve. Bring total volume to 1 L with dH₂O. Filter toremove particulate matter.

7. 1% BSA in PBS: To make a 1× working solution, add 10 g BSA to approx.990 ml PBS buffer, stir to dissolve. Adjust total volume to 1 L with PBSbuffer, filter to remove particulate matter.

8. 50 mM Hepes pH 7.5.

9. GST-Flk1cd purified from sf9 recombinant baculovirus transformation(SUGEN, Inc.).

10. 4% DMSO in dH₂O.

11. 10 mM ATP in dH₂O.

12. 40 mM MnCl₂

13. Kinase Dilution Buffer (KDB): mix 10 ml Hepes (pH 7.5), 1 ml 5MNaCl, 40 μL 100 mM sodium orthovanadate and 0.4 ml of 5% BSA in dH₂Owith 88.56 ml dH₂O.

14. NUNC 96-well V bottom polypropylene plates, Applied ScientificCatalog #AS-72092

15. EDTA: mix 14.12 g ethylenediaminetetraacetic acid (EDTA) to approx.70 ml dH₂O. Add 10 N NaOH until EDTA dissolves. Adjust pH to 8.0. Adjusttotal volume to 100 ml with dH₂O.

16. 1° Antibody Dilution Buffer: mix 10 ml of 5% BSA in PBS buffer with89.5 ml TBST.

17. Anti-phosphotyrosine monoclonal antibody conjugated to horseradishperoxidase (PY99 HRP, Santa Cruz Biotech).

18. 2,2′-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS, Moss,Cat. No. ABST).

19. 10% SDS.

Procedure:

1. Coat Corning 96-well ELISA plates with 2 μg of polyEY peptide insterile PBS as described in step 3 of Materials and Reagents.

2. Remove unbound liquid from wells by inverting plate. Wash once withTBST. Pat the plate on a paper towel to remove excess liquid.

3. Add 100 μl of 1% BSA in PBS to each well. Incubate, with shaking, for1 hr. at room temperature.

4. Repeat step 2.

5. Soak wells with 50 mM HEPES (pH7.5) (150 μl/well).

6. Dilute test compound with dH₂O/4% DMSO to 4 times the desired finalassay concentration in 96-well polypropylene plates.

7. Add 25 μl diluted test compound to ELISA plate. In control wells,place 25 μl of dH₂O/4% DMSO.

8. Add 25 μl of 40 mM MnCl₂ with 4× ATP (2 μM) to each well.

9. Add 25 μl 0.5M EDTA to negative control wells.

10. Dilute GST-Flk1 to 0.005 μg (5 ng)/well with KDB.

11. Add 50 μl of diluted enzyme to each well.

12. Incubate, with shaking, for 15 minutes at room temperature.

13. Stop reaction by adding 50 μl of 250 mM EDTA (pH 8.0).

14. Wash 3× with TBST and pat plate on paper towel to remove excessliquid.

15. Add 100 μl per well anti-phosphotyrosine HRP conjugate, 1:5,000dilution in antibody dilution buffer. Incubate, with shaking, for 90min. at room temperature.

16. Wash as in step 14.

17. Add 100 μl of room temperature ABTS solution to each well.

18. Incubate, with shaking, for 10 to 15 minutes. Remove any bubbles.

19. Stop reaction by adding 20 μl of 10% SDS to each well.

20. Read results on Dynatech MR7000 ELISA reader with test filter at 410nM and reference filter at 630 nM.

PYK2 Bioassay

This assay is used to measure the in vitro kinase activity of HAepitope-tagged full length pyk2 (FL.pyk2-HA) in an ELISA assay.

Materials and Reagents:

1. Corning 96-well Elisa plates.

2. 12CA5 monoclonal anti-HA antibody (SUGEN, Inc.)

3. PBS (Dulbecco's Phosphate-Buffered Saline (Gibco Catalog #450-1300EB)

4. TBST Buffer: for 1 L, mix 8.766 g NaCl, 6.057 g TRIS and 1 ml of 0.1%Triton X-100 in approx. 900 ml dH₂O. Adjust pH to 7.2, bring volume to 1L.

5. Blocking Buffer: for 1 L, mix 100 g 10% BSA, 12.1 g 100 mM TRIS,58.44 g 1M NaCl and 10 mL of 1% TWEEN-20.

6. FL.pyk2-HA from sf9 cell lysates (SUGEN, Inc.).

7. 4% DMSO in MilliQue H₂O.

8. 10 mM ATP in dH₂O.

9. 1M MnCl₂.

10. 1M MgCl₂.

11. 1M Dithiothreitol (DTT).

12. 10× Kinase buffer phosphorylation: mix 5.0 ml 1M Hepes (pH 7.5), 0.2ml 1M MnCl₂, 1.0 ml 1 M MgCl₂, 1.0 ml 10% Triton X-100 in 2.8 ml dH₂O.Just prior to use, add 0.1 ml 1M DTT.

13. NUNC 96-well V bottom polypropylene plates.

14. 500 mM. EDTA in dH₂O.

15. Antibody dilution buffer: for 100 mL, 1 mL 5% BSA/PBS and 1 mL 10%Tween-20 in 88 mL TBS.

16. HRP-conjugated anti-Ptyr PY99), Santa Cruz Biotech Cat. No. SC-7020.

17. ABTS, Moss, Cat. No. ABST-2000.

18. 10% SDS.

Procedure:

1. Coat Corning 96 well ELISA plates with 0.5 μg per well 12CA5 anti-HAantibody in 100 μl PBS. Store overnight at 4° C.

2. Remove unbound HA antibody from wells by inverting plate. Wash platewith dH₂O. Pat the plate on a paper towel to remove excess liquid.

3. Add 150 μl Blocking Buffer to each well. Incubate, with shaking, for30 min at room temperature.

4. Wash plate 4× with TBS-T.

5. Dilute lysate in PBS (1.5 μg lysate/100 μl PBS).

6. Add 100 μl of diluted lysate to each well. Shake at room temperaturefor 1 hr.

7. Wash as in step 4.

8. Add 50 μl of 2× kinase Buffer to ELISA plate containing capturedpyk2-HA.

9. Add 25 μL of 400 μM test compound in 4% DMSO to each well. Forcontrol wells use 4% DMSO alone.

10. Add 25 μL of 0.5 M EDTA to negative control wells.

11. Add 25 μl of 20 μM ATP to all wells. Incubate, with shaking, for 10minutes.

12. Stop reaction by adding 25 μl 500 mM EDTA (pH 8.0) to all wells.

13. Wash as in step 4.

14. Add 100 μL HRP conjugated anti-Ptyr diluted 1:6000 in AntibodyDilution Buffer to each well. Incubate, with shaking, for 1 hr. at roomtemperature.

15. Wash plate 3× with TBST and 1× with PBS.

16. Add 100 μL of ABST solution to each well.

17. If necessary, stop the development reaction by adding 20 μL 10% SDSto each well.

18. Read plate on ELISA reader with test filter at 410 nM and referencefilter at 630 nM.

FGFR1 Bioassay

This assay is used to measure the in vitro kinase activity of FGF1-R inan ELISA assay.

Materials and Reagents:

1. Costar 96-well Elisa plates (Corning Catalog #3369).

2. Poly(Glu-Tyr) (Sigma Catalog #PO275).

3. PBS (Gibco Catalog #450-1300EB)

4. 50 mM Hepes Buffer Solution.

5. Blocking Buffer (5% BSA/PBS).

6. Purified GST-FGFR1 (SUGEN, Inc.)

7. Kinase Dilution Buffer.

-   -   Mix 500 μl 1M Hepes (GIBCO), 20 μl 5% BSA/PBS, 10 μl 100 mM        sodium orthovanadate and 50 μl 5M NaCl.

8. 10 mM ATP

9. ATP/MnCl₂ phosphorylation mix: mix 20 μL ATP, 400 μL 1M MnCl₂ and9.56 ml dH₂O.

10. NUNC 96-well V bottom polypropylene plates (Applied ScientificCatalog #AS-72092).

11. 0.5M EDTA.

12. 0.05% TBST

-   -   Add 500 μL TWEEN to 1 liter TBS.

13. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).

14. Goat anti-rabbit IgG peroxidase conjugate (Biosource, Catalog#ALI0404).

15. ABTS Solution.

16. ABTS/H₂O₂ solution.

Procedure:

1. Coat Costar 96 well ELISA plates with 1 μg per well Poly(Glu, Tyr) in100 μl PBS. Store overnight at 4° C.

2. Wash coated plates once with PBS.

3. Add 150 μL of 5%BSA/PBS Blocking Buffer to each well. Incubate, withshaking, for 1 hr.room temperature.

4. Wash plate 2× with PBS, then once with 50 mM Hepes.

Pat plates on a paper towel to remove excess liquid and bubbles.

5. Add 25 μL of 0.4 mM test compound in 4% DMSO or 4% DMSO alone(controls) to plate.

6. Dilute purified GST-FGFR1 in Kinase Dilution Buffer (5 ng kinase/50ul KDB/well).

7. Add 50 μL of diluted kinase to each well.

8. Start kinase reaction by adding 25 μl/well of freshly preparedATP/Mn++ (0.4 ml 1M MnCl₂, 40 μL 10 mM ATP, 9.56 ml dH₂O), freshlyprepared).

9. This is a fast kinase reaction and must be stopped with 25 μL of 0.5MEDTA in a manner similar to the addition of ATP.

10. Wash plate 4× with fresh TBST.

11. Make up Antibody Dilution Buffer: Per 50 ml: Mix 5 ml of 5% BSA, 250μl of 5% milk and 50 μl of 100 mM sodium vanadate, bring to final volumewith 0.05% TBST.

12. Add 100 μl per well of anti-phosphotyrosine (1:10000 dilution inAbB). Incubate, with shaking for 1 hr. at room temperature.

13. Wash as in step 10.

14. Add 100 μl per well of Biosource Goat anti-rabbit IgG peroxidaseconjugate (1:6000 dilution in ADB). Incubate, with shaking for 1 hr. atroom temperature.

15. Wash as in step 10 and then with PBS to remove bubbles and excessTWEEN.

16. Add 100 μl of ABTS/H₂O₂ solution to each well.

17. Incubate, with shaking, for 10 to 20 minutes.

Remove any bubbles.

18. Read assay on Dynatech MR7000 elisa reader: test filter at 410 nM,reference filtrate 630 nM.

EGFR Biossay

This assay is used to the in vitro kinase activity of FGF1-R in an ELISAassay.

Materials and Reagents:

1. Corning 96-well Elisa plates.

2. SUMO1 monoclonal anti-EGFR antibody. (SUGEN, Inc.).

3. PBS

4. TBST Buffer

5. Blocking Buffer: for 100 ml, mix 5.0 g Carnation Instant Non-fatMilk® with 100 ml of PBS.

6. A431 cell lysate (SUGEN, Inc.).

7. TBS Buffer:

8. TBS+10% DMSO: for 1L, mix 1.514 g TRIS, 2.192 g NaCl and 25 ml DMSO;bring to 1 liter total volume with dH₂O.

9. ATP (Adenosine-5′-triphosphate, from Equine muscle, Sigma Cat. No.A-5394), 1.0 mM solution in dH₂O.

This reagent should be made up immediately prior to use and kept on ice.

10. 1.0 mM MnCl₂.

11. ATP/MnCl₂ phosphorylation mix: to make 10 ml, mix 300 μl of 1 mMATP, 500 μl MnCl₂ and 9.2 ml dH₂O.

Prepare just prior to use, keep on ice.

12. NUNC 96-well V bottom polypropylene plates.

13. EDTA.

14. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).

15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat. No.ALI0404)

16. ABTS.

17. 30% Hydrogen peroxide.

18. ABTS/H₂O₂.

19. 0.2 M HCl.

Procedure:

1. Coat Corning 96 well ELISA plates with 0.5 μg SUMO1 in 100 μl PBS perwell, store overnight at 4° C.

2. Remove unbound SUMO1 from wells by inverting plate to remove liquid.Wash 1× with dH₂O. Pat the plate on a paper towel to remove excessliquid.

3. Add 150 μl of Blocking Buffer to each well. Incubate, with shaking,for 30 min. at room temperature.

4. Wash plate 3× with deionized water, then once with TBST. Pat plate ona paper towel to remove excess liquid and bubbles.

5. Dilute lysate in PBS (7 μg lysate/100 μl PBS).

6. Add 100 μl of diluted lysate to each well. Shake at room temperaturefor 1 hr.

7. Wash plates as in 4, above.

8. Add 120 μl TBS to ELISA plate containing captured EGFR.

9. Dilute test compound 1:10 in TBS, place in well

10. Add 13.5 μl diluted test compound to ELISA plate. To control wells,add. 13.5 μl TBS in 10% DMSO.

11. Incubate, with shaking, for 30 minutes at room temperature.

12. Add 15 μl phosphorylation mix to all wells except negative controlwell. Final well volume should be approximately 150μl with 3 FM ATP/5 mMMnCl₂ final concentration in each well. Incubate with shaking for 5minutes.

13. Stop reaction by adding 16.5 μl of EDTA solution while shaking.Shake for additional 1 min.

14. Wash 4× with deionized water, 2× with TBST.

15. Add 100 μl anti-phosphotyrosine (1:3000 dilution in TBST) per well.Incubate, with shaking, for 30-45 min. at room temperature.

16. Wash as in 4, above.

17. Add 100 μl Biosource Goat anti-rabbit IgG peroxidase conjugate(1:2000 dilution in TBST) to each well. Incubate with shaking for 30min. at room temperature.

18. Wash as in 4, above.

19. Add 100 μl of ABTS/H₂O₂ solution to each well.

20. Incubate 5 to 10 minutes with shaking. Remove any bubbles.

21. If necessary, stop reaction by adding 100 μl 0.2 M HCl per well.

22. Read assay on Dynatech MR7000 ELISA reader: test filter at 410 nM,reference filter at 630 nM.

PDGFR Bioassay

This assay is used to the in vitro kinase activity of FGF1-R in an ELISAassay.

Materials and Reagents:

1. Corning 96-well Elisa plates

2. 28D4C10 monoclonal anti-PDGFR antibody (SUGEN, Inc.).

3. PBS.

4. TBST Buffer.

5. Blocking Buffer (same as for EGFR bioassay). 6. PDGFR-β expressingNIH 3T3 cell lysate (SUGEN, Inc.).

7. TBS Buffer.

8. TBS+10% DMSO.

9. ATP.

10. MnCl₂.

11. Kinase buffer phosphorylation mix: for 10 ml, mix 250 μl 1M TRIS,200 μl 5M NaCl, 100 μl 1M MnCl₂ and 50 μl 100 mM Triton X-100 in enoughdH₂O to make 10 ml.

12. NUNC 96-well V bottom polypropylene plates.

13. EDTA.

14. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN,Inc.).

15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat. No.ALI0404).

16. ABTS.

17. Hydrogen peroxide, 30% solution.

18. ABTS/H₂O₂.

19. 0.2 M HCl.

Procedure:

1. Coat Corning 96 well ELISA plates with 0.5 μg 28D4C10 in 100 μl PBSper well, store overnight at 4° C.

2. Remove unbound 28D4C10 from wells by inverting plate to removeliquid. Wash 1× with dH₂O. Pat the plate on a paper towel to removeexcess liquid.

3. Add 150 μl of Blocking Buffer to each well. Incubate for 30 min. atroom temperature with shaking.

4. Wash plate 3× with deionized water, then once with TBST. Pat plate ona paper towel to remove excess liquid and bubbles.

5. Dilute lysate in HNTG (10 μg lysate/100 μl HNTG).

6. Add 100 μl of diluted lysate to each well. Shake at room temperaturefor 60 min.

7. Wash plates as described in Step 4.

8. Add 80 μl working kinase buffer mix to ELISA plate containingcaptured PDGFR.

9. Dilute test compound 1:10 in TBS in 96-well polypropylene plates.

10. Add 10 μl diluted test compound to ELISA plate. To control wells,add 10 μl TBS+10% DMSO. Incubate with shaking for 30 minutes at roomtemperature.

11. Add 10 μl ATP directly to all wells except negative control well(final well volume should be approximately 100 μl with 20 μM ATP in eachwell.) Incubate 30 minutes with shaking.

12. Stop reaction by adding 10 μl of EDTA solution to each well.

13. Wash 4× with deionized water, twice with TBST.

14. Add 100 μl anti-phosphotyrosine (1:3000 dilution in TBST) per well.Incubate with shaking for 30-45 min. at room temperature.

15. Wash as in Step 4.

16. Add 100 μl Biosource Goat anti-rabbit IgG peroxidase conjugate(1:2000 dilution in TBST) to each well. Incubate with shaking for 30min. at room temperature.

17. Wash as in Step 4.

18. Add 100 μl of ABTS/H₂O₂ solution to each well.

19. Incubate 10 to 30 minutes with shaking. Remove any bubbles.

20. If necessary stop reaction with the addition of 100 μl 0.2 M HCl perwell.

21. Read assay on Dynatech MR7000 ELISA reader with test filter at 410nM and reference filter at 630 nM.

Cellular HER-2 Kinase Assay

This assay is used to measure HER-2 kinase activity in whole cells in anELISA format.

Materials and Reagents:

1. DMEM (GIBCO Catalog #11965-092).

2. Fetal Bovine Serum (FBS, GIBCO Catalog #16000-044), heat inactivatedin a water bath for 30 min. at 56° C.

3. Trypsin (GIBCO Catalog #25200-056).

4. L-Glutamine (GIBCO Catalog #25030-081)

5. HEPES (GIBCO Catalog #15630-080).

6. Growth Media

-   -   Mix 500ml DMEM, 55 ml heat inactivated FBS, 10 ml HEPES and 5.5        ml L-Glutamine.

7. Starve Media

-   -   Mix 500 ml DMEM, 2.5 ml heat inactivated FBS, 10 ml HEPES and        5.5 ml L-Glutamine.

8. PBS.

9. Flat Bottom 96-well Tissue Culture Micro Titer Plates (CorningCatalog #25860).

10. 15 cm Tissue Culture Dishes (Corning Catalog #08757148).

11. Corning 96-well ELISA Plates.

12. NUNC 96-well V bottom polypropylene plates.

13. Costar Transfer Cartridges for the Transtar 96 (costar Catalog#7610).

14. SUMO 1: monoclonal anti-EGFR antibody (SUGEN, Inc.).

15. TBST Buffer.

16. Blocking Buffer : 5% Carnation Instant Milk® in PBS.

17. EGF Ligand: EGF-201, Shinko American, Japan.

-   -   Suspend powder in 100 uL of 10 mM HCl. Add 100 uL 10 mM NaOH.        Add 800 uL PBS and transfer to an Eppendorf tube, store at        −20° C. until ready to use.

18. HNTG Lysis Buffer

-   -   For Stock 5× HNTG, mix 23.83 g Hepes, 43.83 g NaCl, 500 ml        glycerol and 100 ml Triton X-100 and enough dH₂O to make 1 L of        total solution.    -   For 1× HNTG*, mix 2 ml HNTG, 100 μL 0.1M Na₃VO₄, 250 μL 0.2M        Na₄P₂O₇ and 100 uL EDTA.

19. EDTA.

20. Na₃VO₄. To make stock solution, mix 1.84 g Na₃VO₄ with.90 ml dH₂O.Adjust pH to 10. Boil in microwave for one minute (solution becomesclear). Cool to room temperature. Adjust pH to 10. Repeatheating/cooling cycle until pH remains at 10.

21. 200 mM Na₄P₂O₇.

22. Rabbit polyclonal antiserum specific for phosphotyrosine (anti-Ptyrantibody, SUGEN, Inc.).

23. Affinity purified antiserum, goat anti-rabbit IgG antibody,peroxidase conjugate (Biosource Cat. #ALI0404),

24. ABTS Solution.

25. 30% Hydrogen peroxide solution.

26. ABTS/H₂O₂.

27. 0.2 M HCl.

Procedure:

1. Coat Corning 96 well ELISA plates with SUMO1 at 1.0 ug per well inPBS, 100 ul final volume/well. Store overnight at 4° C.

2. On day of use, remove coating buffer and wash plate 3 times with dH₂Oand once with TBST buffer. All washes in this assay should be done inthis manner, unless otherwise specified.

3. Add 100 ul of Blocking Buffer to each well. Incubate plate, withshaking, for 30 min. at room temperature. Just prior to use, wash plate.

4. Use EGFr/HER-2 chimera/3T3-C7 cell line for this assay.

5. Choose dishes having 80-90% confluence. Collect cells bytrypsinization and centrifuge at 1000 rpm at room temperature for 5 min.

6. Resuspend cells in starve medium and count with trypan blue.Viability above 90% is required. Seed cells in starve medium at adensity of 2,500 cells per well, 90 ul per well, in a 96 well microtiterplate. Incubate seeded cells overnight at 37° under 5% CO₂.

7. Start the assay two days after seeding.

8. Test compounds are dissolved in 4% DMSO. Samples are then furtherdiluted directly on plates with starve-DMEM. Typically, this dilutionwill be 1:10 or greater. All wells are then transferred to the cellplate at a further 1:10 dilution (10 μl sample and media into 90 μl ofstarve media. The final DMSO concentration should be 1% or lower. Astandard serial dilution may also be used.

9. Incubate under 5% CO₂ at 37° C. for 2 hours. 10. Prepare EGF ligandby diluting stock EGF (16.5 uM) in warm DMEM to 150 nM. 11. Preparefresh HNTG* sufficient for 100 ul per well; place on ice.

12. After 2 hour incubation with test compound, add prepared EGF ligandto cells, 50 ul per well, for a final concentration of 50 nM. Positivecontrol wells receive the same amount of EGF. Negative controls do notreceive EGF. Incubate at 37° C. for 10 min.

13. Remove test compound, EGF, and DMEM. Wash cells once with PBS.

14. Transfer HNTG* to cells, 100 ul per well. Place on ice for 5minutes. Meanwhile, remove blocking buffer from ELISA plate and wash.

15. Scrape cells from plate with a micropipettor and homogenize cellmaterial by repeatedly aspirating and dispensing the HNTG* lysis buffer.Transfer lysate to a coated, blocked, washed ELISA plate. Or, use aCostar transfer cartridge to transfer lysate to the plate.

16. Incubate, with shaking, at room temperature for 1 hr.

17. Remove lysate, wash. Transfer freshly diluted anti-Ptyr antibody(1:3000 in TBST) to ELISA plate, 100 ul per well.

18. Incubate, with shaking, at room temperature, for 30 min.

19. Remove anti-Ptyr antibody, wash. Transfer freshly diluted BIOSOURCEantibody to ELISA plate(1:8000 in TBST, 100 ul per well).

20. Incubate, with shaking, at room temperature for 30 min.

21. Remove BIOSOURCE antibody, wash. Transfer freshly prepared ABTS/H₂O₂solution to ELISA plate, 100 ul per well.

22. Incubate, with shaking, for 5-10 minutes. Remove any bubbles.

23. Stop reaction with the addition of 100 ul of 0.2M HCl per well.

24. Read assay on Dynatech MR7000 ELISA reader with test filter set at410 nM and reference filter at 630 nM.

CDK2/Cyclin A Assay

This assay is used to measure the in vitro serine/threonine kinaseactivity of human cdk2/cyclin A in a Scintillation Proximity Assay(SPA).

Materials and Reagents.

1. Wallac 96-well polyethylene terephthalate (flexi) plates (WallacCatalog #1450-401).

2. Amersham Redivue [γ³³P] ATP (Amersham catalog #AH 9968).

3. Amersham streptavidin coated polyvinyltoluene SPA beads (Amershamcatalog #RPNQ0007). The beads should be reconstituted in PBS withoutmagnesium or calcium, at 20 mg/ml.

4. Activated cdk2/cyclin A enzyme complex purified from Sf9 cells(SUGEN, Inc.).

5. Biotinylated peptide substrate (Debtide). Peptide biotin-X-PKTPKKAKKLis dissolved in dH₂O at a concentration of 5 mg/ml.

6. Peptide/ATP Mixture: for 10 ml, mix 9.979 ml dH₂O, 0.00125 ml “cold”ATP, 0.010 ml Debtide and 0.010 ml γ³³P ATP. The ultimate concentrationper well will be 0.5 μM “cold” ATP, 0.1 μg Debtide and 0.2 μCi γ³³P ATP.

7. Kinase buffer: for 10 ml, mix 8.85 ml dH₂O, 0.625 ml TRIS(pH 7.4),0.25 ml 1M MgCl₂, 0.25 ml 10% NP40 and 0.025 ml 1M DTT, added fresh justprior to use.,

8. 10 mM ATP in dH₂O. P 9. 1M Tris, pH adjusted to 7.4 with HCl.

10. 1M MgCl₂.

11. 1M DTT.

12. PBS (Gibco Catalog #14190-144).

13. 0.5M EDTA.

14. Stop solution: For 10 ml, mix 9.25 ml PBS, 0.005 ml 100 mM ATP, 0.1ml 0.5 M EDTA, 0.1 ml 10% Triton X-100 and 1.25 ml of 20 mg/ml SPAbeads.

Procedure:

1. Prepare solutions of test compounds at 5× the desired finalconcentration in 5% DMSO. Add 10 ul to each well. For negative controls,use 10 ul 5% DMSO alone in wells.

2. Dilute 5 μl of cdk2/cyclin A solution with 2.1 ml 2× kinase buffer.

3. Add 20 ul enzyme to each well.

4. Add 10 μL of 0.5 M EDTA to the negative control wells.

5. To start kinase reaction, add 20 μL of peptide/ATP mixture to eachwell. Incubate for 1 hr. without shaking.

6. Add 200 μl stop solution to each well.

7. Hold at least 10 min.

8. Spin plate at approx. 2300 rpm for 3-5 min.

9. Count plate using Trilux or similar reader.

Met Transportation Assay

This assay is used to measure phosphotyrosine levels on a poly(glutamicacid:tyrosine (4:1)) substrate as a means for identifyingagonists/antagonists of met transphosphorylation of the substrate.

Materials and Reagents:

1. Corning 96-well Elisa plates, Corning Catalog #25805-96.

2. Poly(glu, tyr) 4:1, Sigma, Cat. No; P 0275.

3. PBS, Gibco Catalog #450-1300EB

4. 50 mM HEPES

5. Blocking Buffer: Dissolve 25 g Bovine Serum Albumin, Sigma Cat. NoA-7888, in 500 ml PBS, filter through a 4 μm filter.

6. Purified GST fusion protein containing the Met kinase domain, Sugen,Inc.

7. TBST Buffer.

8. 10% aqueous (MilliQue H₂O) DMSO.

9. 10 mM aqueous (dH₂O) Adenosine-5′-triphosphate, Sigma 35 Cat. No.A-5394.

10. 2× Kinase Dilution Buffer: for 100 ml, mix 10 mL 1M HEPES at pH 7.5with 0.4 mL 5% BSA/PBS, 0.2 mL 0.1 M sodium orthovanadate and 1 mL 5Msodium chloride in 88.4 mL dH₂O.

11. 4× ATP Reaction Mixture: for 10 mL, mix 0.4 mL 1 M manganesechloride and 0.02 mL 0.1 M ATP in 9.56 mL dH₂O.

12. 4× Negative Controls Mixture: for 10 mL, mix 0.4 mL 1 M manganesechloride in 9.6 mL dH₂O.

13. NUNC 96-well V bottom polypropylene plates, Applied ScientificCatalog #S-72092

14. 500 mM EDTA.

15. Antibody Dilution Buffer: for 100 mL, mix 10 mL 5% BSA/PBS, 0.5 mL5% Carnation Instant Milk® in PBS and 0.1 mL 0.1 M sodium orthovanadatein 88.4 mL TBST.

16. Rabbit polyclonal antophosphotyrosine antibody, Sugen, Inc.

17. Goat anti-rabbit horseradish peroxidase conjugated antibody,Biosource, Inc.

18. ABTS Solution: for 1 L, mix 19.21 g citric acid, 35.49;g Na₂HPO₄ and500 mg ABTS with sufficient dH₂O to make 1 L.

19. ABTS/H₂O₂: mix 15 mL ABST solution with 2 μL H₂O₂ five minutesbefore use.

20. 0.2 M HCl

Procedure:

1. Coat ELISA plates with 2 μg Poly(Glu-Tyr) in 100 μL PBS, storeovernight at 4° C.

2. Block plate with 150 μL of 5% BSA/PBS for 60 min.

3. Wash plate twice with PBS, once with 50 mM Hepes buffer pH 7.4.

4. Add 50 μl of the diluted kinase to all wells. (Purified kinase isdiluted with Kinase Dilution Buffer. Final concentration should be 10ng/well.)

5. Add 25 μL of the test compound (in 4% DMSO) or DMSO alone (4% indH₂O) for controls to plate.

6. Incubate the kinase/compound mixture for 15 minutes.

7. Add 25 μL of 40 mM MnCl₂ to the negative control wells.

8. Add 25 μL ATP/MnCl₂ mixture to the all other wells (except thenegative controls). Incubate for 5 min.

9. Add 25 μL 500 mM EDTA to stop reaction.

10. Wash plate 3× with TBST.

11. Add 100 μl rabbit polyclonal anti-Ptyr diluted 1:10,000 in AntibodyDilution Buffer to each well. Incubate, with shaking, at roomtemperature for one hour.

12. Wash plate 3× with TBST.

13. Dilute Biosource HRP conjugated anti-rabbit antibody 1:6,000 inAntibody Dilution buffer. Add 100 μL per well and incubate at roomtemperature, with shaking, for one hour.

14. Wash plate 1× with PBS.

15. Add 100 μl of ABTS/H₂O₂ solution to each well.

16. If necessary, stop the development reaction with the addition of 100μl of 0.2M HCl per well.

17. Read plate on Dynatech MR7000 elisa reader with the test filter at410 nM and the reference filter at 630 nM.

IGF-1 Transphosphorylation Assay

This assay is used to measure the phosphotyrosine level in poly(glutamicacid:tyrosine)(4:1) for the identification of agonists/antagonists ofgst-IGF-1 transphosphorylation of a substrate.

Materials and Reagents:

1. Corning 96-well Elisa plates.

2. Poly (Glu-tyr) (4:1), Sigma Cat. No. P 0275.

3. PBS, Gibco Catalog #450-1300EB.

4. 50 mM HEPES

5. TBB Blocking Buffer: for 1 L, mix 100 g BSA, 12.1 gTRIS (pH 7.5),58.44 g sodium chloride and 10 mL 1% TWEEN-20.

6. Purified GST fusion protein containing the IGF-1 kinase domain(Sugen, Inc.)

7. TBST Buffer: for 1 L, mix 6.057 g Tris, 8.766 g sodium chloride and0.5 ml TWEEN-20 with enough dH₂O to make 1 liter.

8. 4% DMSO in Milli-Q H₂O.

9. 10 mM ATP in dH₂O.

10. 2× Kinase Dilution Buffer: for 100 mL, mix 10 mL 1 M HEPES (pH 7.5),0.4 mL 5% BSA in dH₂O, 0.2 mL 0.1 M sodium orthovanadate and 1 mL 5. Msodium chloride with enough dH₂O to make 100 mL.

11. 4× ATP Reaction Mixture: for 10 mL, mix 0.4 mL 1 M MnCl₂ and 0.008mL 0.01 M ATP and 9.56 mL dH₂O.

12.4× Negative Controls Mixture: mix 0.4 mL 1 M manganese chloride in9.60 mL dH₂O.

13. NUNC 96-well V bottom polypropylene plates.

14. 500 mM EDTA in dH₂O.

15. Antibody Dilution Buffer: for 100 mL, mix 10 mL 5% BSA in PBS, 0.5mL 5% Carnation Instant Non-fat Milk® in PBS and 0.1 mL 0.1 M sodiumorthovanadate in 88.4 mL TBST.

16. Rabbit Polyclonal antiphosphotyrosine antibody, Sugen, Inc.

17. Goat anti-rabbit HRP conjugated antibody, Biosource.

18. ABTS Solution.

20. ABTS/H₂O₂: mix 15 mL ABTS with 2 μL H₂O₂ 5 minutes before using.

21. 0.2 M HCl in dH₂O.

Procedure:

1. Coat ELISA plate with 2.0 μg/well Poly(Glu, Tyr) 4:1 (Sigma P0275) in100 μl PBS. Store plate overnight at 4° C.

2. ash plate once with PBS.

3. Add 100 μl of TBB Blocking Buffer to each well.

Incubate plate for l hour with shaking at room temperature.

4. Wash plate once with PBS, then twice with 50 mM Hepes buffer pH 7.5.

5. Add 25 μL of test compound in 4% DMSO (obtained by diluting a stocksolution of 10 mM test compound in 100% DMSO with dH₂O) to plate.

6. Add 10.0 ng of gst-IGF-1 kinase in 50 μl Kinase Dilution Buffer) toall wells.

7. Start kinase reaction by adding 25 μl 4× ATP Reaction Mixture to alltest wells and positive control wells. Add 25 μl 4× Negative ControlsMixture to all negative control wells. Incubates for 10 minutes withshaking at room temperature.

8. Add 25 μl 0.5M EDTA (pH 8.0) to all wells.

9. Wash plate 4× with TBST Buffer.

10. Add rabbit polyclonal anti-phosphotyrosine antisera at a dilution of1:10,000 in 100 μl Antibody Dilution Buffer to all wells. Incubate, withshaking, at room temperature for 1 hour.

11. Wash plate as in step 9.

12. Add 100 μl Biosource anti-rabbit HRP at a dilution of 1:10,000 inAntibody dilution buffer to all wells. Incubate, with shaking, atroomtemperature for 1 hour.

13. Wash plate as in step 9, follow with one wash with PBS to reducebubbles and excess Tween-20.

14. Develop by adding 100μl/well ABTS/H₂O₂ to each well.

15. After about 5 minutes, read on ELISA reader with test filter at 410nm and referenced filter at 630 nm.

BrdU Incorporation Assays

The following assays use cells engineered to express a selected receptorand then evaluate the effect of a compound of interest on the activityof ligand-induced DNA synthesis by determining BrdU incorporation intothe DNA.

The following materials, reagents and procedure are general to each ofthe following BrdU incorporation assays. Variances in specific assaysare noted.

Materials and Reagents:

1. The appropriate ligand.

2. The appropriate engineered cells.

3. BrdU Labeling Reagent: 10 mM, in PBS (pH 7.4).(Boehringer Mannheim,Germany).

4. FixDenat: fixation solution (ready to use)(Boehringer Mannheim,Germany).

5. Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase(Boehringer Mannheim, Germany).

6. TMB Substrate Solution: tetramethylbenzidine (TMB, BoehringerMannheim, Germany).

7. PBS Washing Solution: 1× PBS, pH 7.4.

8. Albumin, Bovine (BSA), fraction V powder (Sigma Chemical Co., USA).

General Procedure:

1. Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln in DMEM, in a96 well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

2. After 24 hours, the cells are washed with PBS, and then areserum-starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24hours.

3. On day 3, the appropriate ligand and the test compound are added tothe cells simultaneously. The negative control wells receive serum freeDMEM with 0.1% BSA only; the positive control cells receive the ligandbut no test compound. Test compounds are prepared in serum free DMEMwith ligand in a 96 well plate, and serially diluted for 7 testconcentrations.

4. After 18 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU(final concentration=10 μM) for 1.5 hours.

5. After incubation with labeling reagent, the medium is removed bydecanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

6. The FixDenat solution is thoroughly removed by decanting and tappingthe inverted plate on a paper towel. Milk is added (5% dehydrated milkin PBS, 200 μl/well) as a blocking solution and the plate is incubatedfor 30 minutes at room temperature on a plate shaker.

7. The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution (1:200 dilution in PBS, 1%BSA) is added (50 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

8. The antibody conjugate is thoroughly removed by decanting and rinsingthe wells 5 times with PBS, and the plate is dried by inverting andtapping on a paper towel.

9. TMB substrate solution is added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color development issufficient for photometric detection.

10. The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

EGF-Induced BrdU Incorporation Assay

Materials and Reagents:

1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).

2. 3T3/EGFRc7.

EGF-Induced Her-2-driven BrdU Incorporation Assay

Materials and Reagents:

1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).

2. 3T3/EGFr/Her2/EGFr (EGFr with a Her-2 kinase domain).

EGF-Induced Her-4-Driven BrdU Incorporation Assay

Materials and Reagents:

1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).

2. 3T3/EGFr/Her4/EGFr (EGFr with a Her-4 kinase domain).

PDGF-Induced BrdU Incorporation Assay

Materials and Reagents:

1. Human PDGF B/B (Boehringer Mannheim, Germany).

2. 3T3/EGFRc7.

FGF-Induced BrdU Incorporation Assay

Materials and Reagents:

1. Human FGF2/bFGF (Gibco BRL, USA).

2. 3T3c7/EGFr

IGF1-Induced BrdU Incorporation Assay

Materials and Reagents:

1. Human, recombinant (G511, Promega Corp., USA)

2. 3T3/IGF1r.

Insulin-Induced BrdU Incorporation Assay

Materials and Reagents:

1. Insulin, crystalline, bovine, Zinc (13007, Gibco BRL, USA).

2. 3T3/H25.

HGF-Induced BrdU Incorporation Assay

Materials and Reagents: P 1. Recombinant human HGF (Cat. No. 249-HG, R&DSystems, Inc. USA).

2. BxPC-3 cells (ATCC CRL-1687).

Procedure:

1. Cells are seeded at 9000 cells/well in RPMI 10% FBS in a 96 wellplate. Cells are incubated overnight at 37° C. in 5% CO₂.

2. After 24 hours, the cells are washed with PBS, and then are serumstarved in 100 μl serum-free medium (RPMI with 0.1% BSA) for 24 hours.

3. On day 3, 25 μl containing ligand (prepared at 1 μg/ml in RPMI with0.1% BSA; final HGF conc. is 200 ng/ml) and test compounds are added tothe cells. The negative control wells receive 25 μl serum-free RPMI with0.1% BSA only; the positive control cells receive the ligand (HGF) butno test compound. Test compounds are prepared at 5 times their finalconcentration in serum-free-RPMI with ligand in a 96 well plate, andserially diluted to give 7 test concentrations. Typically, the highestfinal concentration of test compound is 100 μM, and 1:3 dilutions areused (i.e. final test compound concentration range is 0.137-100 μM).

4. After 18 hours of ligand activation, 12.5 μl of diluted BrdU labelingreagent (1:100 in RPMI, 0.1% BSA) is added to each well and the cellsare incubated with BrdU (final concentration is 10 μM) for 1 hour.

5. Same as General Procedure.

6. Same as General Procedure.

7. The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%BSA) is added (100 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

8. Same as General Procedure.

9. Same as General Procedure.

10. Same as General Procedure.

HUV-EC-C Assay

This assay is used to measure a compound's activity against PDGF-R,FGF-R, VEGF, aFGF or Flk-1/KDR, all of which are naturally expressed byHUV-EC cells.

Day 0

1. Wash and trypsinize HUV-EC-C cells (human umbilical vein endothelialcells, (American Type Culture Collection, catalogue no. 1730 CRL). Washwith Dulbecco's phosphate-buffered saline (D-PBS, obtained from GibcoBRL, catalogue no. 14190-029) 2 times at about 1 ml/cm² of tissueculture flask. Trypsinize with 0.05% trypsin-EDTA in non-enzymatic celldissociation solution (Sigma Chemical Company, catalogue no. C-1544).The 0.05% trypsin is made by diluting 0.25% trypsin/l mM EDTA (Gibco,catalogue no. 25200-049) in the cell dissociation solution. Trypsinizewith about 1 ml/25-30 cm² of tissue culture flask for about 5 minutes at37° C. After-cells have detached from the flask, add an equal volume ofassay medium and transfer to a 50 ml sterile centrifuge tube (FisherScientific, catalogue no. 05-539-6).

2. Wash the cells with about 35 ml assay medium in the 50 ml sterilecentrifuge tube by adding the assay medium, centrifuge for 10 minutes atapproximately 200×g, aspirate the supernatant, and resuspend with 35 mlD-PBS. Repeat the wash two more times with D-PBS, resuspend the cells inabout 1 ml assay medium/15 cm² of tissue culture flask. Assay mediumconsists of F12K medium (Gibco BRL, catalogue no. 21127-014) and 0.5%heat-inactivated fetal bovine serum. Count the cells with a CoulterCounter® (Coulter Electronics, Inc.) and add assay medium to the cellsto obtain a concentration of 0.8-1.0×10⁵ cells/ml.

3. Add cells to 96-well flat-bottom plates at 100 μl/well or 0.8-1.0×10⁴cells/well, incubate 24 h at 37° C., 5% CO₂.

Day 1

1. Make up two-fold test compound titrations in separate 96-well plates,generally 50 μM on down to 0 μM. Use the same assay medium as mentionedin day 0, step 2 above. Titrations are made by adding 90 μl/well of testcompound at 200 μM (4× the final well concentration) to the top well ofa particular plate column. Since the stock test compound is usually 20mM in DMSO, the 200 μM drug concentration contains 2% DMSO.

A diluent made up to 2% DMSO in assay medium (F12K+0.5% fetal bovineserum) is used as diluent for the test compound titrations in order todilute the test compound but keep the DMSO concentration constant. Addthis diluent to the remaining wells in the column at 60 μl/well. Take 60μl from the 120 μl of 200 μM test compound dilution in the top well ofthe column and mix with the 60 μl in the second well of the column. Take60 μl from this well and mix with the 60 μl in the third well of thecolumn, and so on until two-fold titrations are completed. When thenext-to-the-last well is mixed, take 60 μl of the.120 μl in this welland discard it. Leave the last well with 60 μl of DMSO/media diluent asa non-test compound-containing control. Make 9 columns of titrated testcompound, enough for triplicate wells each for: (1) VEGF (obtained fromPepro Tech Inc., catalogue no. 100-200, (2) endothelial cell growthfactor (ECGF) (also known as acidic fibroblast growth factor, or aFGF)(obtained from Boehringer Mannheim Biochemica, catalogue no. 1439 600),or, (3) human PDGF B/B (1276-956, Boehringer Mannheim, Germany) andassay media control. ECGF comes as a preparation with sodium heparin.

2. Transfer 50 μl/well of the test compound dilutions to the 96-wellassay plates containing the 0.8-1.0×10⁴ cells/100 μl/well of theHUV-EC-C cells from day 0 and incubate ˜2 h at 37° C., 5% CO₂.

3. In triplicate, add 50 μl/well of 80 μg/ml VEGF, 20 ng/ml ECGF, ormedia control to each test compound condition. As with the testcompounds, the growth factor concentrations are 4× the desired finalconcentration. Use the assay media from day 0 step 2 to make theconcentrations of growth factors. Incubate approximately 24 hours at 37°C., 5% CO₂. Each well will have 50 μl test compound dilution, 50 μlgrowth factor or media, and 100 μl cells, which calculates to 200μl/well total. Thus the 4× concentrations of test compound and growthfactors become 1× once everything has been added to the wells.

Day 2

1. Add ³H-thymidine (Amersham, catalogue no. TRK-686) at 1 μCi/well (10μl/well of 100 μCi/ml solution made up in RPMI media+10%heat-inactivated fetal bovine serum) and incubate ˜24 h at 37° C., 5%CO₂. RPMI is obtained from Gibco BRL, catalogue no. 11875-051.

Day 3

1. Freeze plates overnight at −20° C.

Day 4

Thaw plates and harvest with a 96-well plate harvester (Tomtec Harvester96®) onto filter mats (Wallac, catalogue no. 1205-401), read counts on aWallac Betaplate™ liquid scintillation counter.

TABLE 3 shows the results of biological testing of some exemplarycompounds of this invention. The results are reported in terms of IC₅₀,the micromolar (μM) concentration of the compound being tested whichcauses a 50% change in the activity of the target PKT compared to theactivity of the PTK in a control to which no test compound has beenadded. Specifically, the results shown indicate the concentration of atest compound needed to cause a 50%reduction of the activity of thetarget PTK. Bioassays which have been or can be used to evaluatecompounds are described in detail below. TABLE 3 bio bio bio bio cellHer2 bio flkGST FGFR1 PDGF EGF EGF Kinase cdk2spa pyk2 IC50 IC50 IC50IC50 IC50 IC50 C50 IC50 Example (μM) (μM) (μM) (μM) (μM) (μM) (μM) (μM)1 57.68 15.16 >100 >100 >100 >100 2 >100 >100 >100 >100 3 9.859.62 >100 >100 >100 >100 4 3.57 >20 >100 >100 >100 >100 5 8.316.06 >100 >100 >100 >100 6 4.04 >100 3.26 7.82 2.43 7 7.74 >100 5.079.8 4.24 8 12.1 >100 51.34 20.08 5.5 9 0.96 >100 >100 >100 16.38 105.72 >100 94.04 15.86 8.06 11 9.77 >100 >100 >100 >100 12 >20 21.46 >10027.73 13 >20 81.92 8.17 2.66 14 13.01 42.41 >100 66.02 15 >20 >100 >10098.61 16 >20 98.06 >100 23.32 17 8.25 2.47 94.35 0.83 11.47 15.94 >10 182.67 2.57 9.23 4.99 19 7.5 6.86 34.18 8.37 20 11.53 >100 41.16 8 217.18 >100 40.34 27.69 22 >20 >100 >100 87.67 23 >20 >100 36.64 4.0524 >100 16.84 5.31 25 12.55 >100 23.48 7.9 26 16.03 66.87 34.67 10.0427 >100 26.5 3.91 28 4.5 71.27 53.66 2.67 29 10.12 >100 26.72 3.98 309.4 >100 18.69 4.1 31 >50 >100 9.83 47.19 32 45.74 5.94 >100 >10034 >50 >100 >100 >100 35 >20 >100 80.4 54.14 36 >20 >100 >100 >100 370.22 3.06 10.78 9.84 1.4 38 4.17 3.06 6.04 8.97 2.16 39 3.38 4.69 3.6714.54 3.53 40 4.5 7.9 6.52 6.27 42 0.1 0.12 11.95 74.55 20.43 43 1.128.38 >100 37.33 53.37 44 <0.05 0.02 20.73 67.46 6.99 45 1.71 >100 >10029.95 >100 46 30.62 6.18 >100 >100 >100 47 0.08 1.56 0.06 11.42 41.548.4 >20 1.05 48 0.006 0.3 <0.78 17.88 21.58 7.93 0.09 49 <0.78 >10043.86 >100 50 <0.78 >100 20.34 >100 51 0.006 1.66 0.01 18.1 21.61 23.2416.69 0.35 52 0.08 1.26 <0.78 12.53 >100 >100 10.66 0.45 53<0.78 >100 >100 >100 54 1.98 <0.78 23.88 9.76 7.02 55 0.27 0.53 6.0335.99 77.82 56 2.32 3.19 >100 10.03 7.11 57 0.06 7.98 >100 9.97 6.94 5821.14 >100 >100 >100 59 <0.78 >100 >100 >100 60 <0.78 >100 >100 >100 61<0.78 >100 >100 >100 62 8.00 8.32 >100 >100 >100 63 0.21 <0.788.59 >100 >100 64 0.55 <0.78 30.49 >100 >100 65 0.37 <0.05 >100 74.3615.97 66 <0.05 >100 11.84 2.76 67 0.39 24.77 31.38 19.79 2.56 68 1.160.03 >100 23.52 34.13 69 0.3 56.55 >100 97.54 >100 70 0.09 1.50 0.003010.57 6.42 7.99 12.62 0.63 71 15.21 22.5 >100 9.91 72 6.06 10.54 >10039.94 9.65 73 5.95 14.12 >100 39.5 8.59 74 1.2 0.09 46.75 >100 75 2.761.55 >100 >100 76 3.33 19.18 5.11 3.01 77 0.49 25.01 >100 >100 78 1.9470.62 9.33 4.25 79 1.49 >100 27.39 >100 80 0.13 4.29 0.001 >100 50.1917.19 0.28 81 0.21 0.18 >100 >100 82 2.03 7.69 6.88 >100 >100 0.31 830.34 0.41 9.46 2.18 86.9 0.008 84 1.38 12.51 67.2 5.86 0.006 85 0.2 0.82.59 >100 3.76 86 1.45 1.3 19.6 41.8 >100 3.58 87 3.27 7.56 6.46 >1009.1 0.17 88 0.35 1.18 8.06 2.36 >100 0.09 89 7.84 47.58 8.53 9.67 15.97115 7.3 7.48 >100 >100 0.006 116 >20 >100 >100 >100 <0.0005 117 0.9112.9 >100 >100 0.006 118 1.93 1.2 >100 >100 0.002 119 1.3861.63 >100 >100 <0.0005In Vivo Animal ModelsXenograft Animal Models

The ability of human tumors to grow as xenografts in athymic mice (e.g.,Balb/c, nu/nu) provides a useful in vivo model for studying thebiological response to therapies for human tumors. Since the firstsuccessful xenotransplantation of human tumors into athymic mice,.(Rygaard and Povlsen, 1969, Acta Pathol. Microbial. Scand. 77:758-760),many different human tumor cell lines (e.g., mammary, lung,genitourinary, gastro-intestinal, head and neck, glioblastoma, bone, andmalignant melanomas) have been transplanted and successfully grown innude mice. The following assays may be used to determine the level ofactivity, specificity and effect of the different compounds of thepresent invention. Three general types of assays are useful forevaluating compounds: cellular/catalytic, cellular/biological and invivo. The object of the cellular/catalytic assays is to determine theeffect of a compound on the ability of a TK to phosphorylate tyrosineson a known substrate in a cell. The object of the cellular/biologicalassays is to determine the effect of a compound on the biologicalresponse stimulated by a TK in a cell. The object of the in vivo assaysis to determine the effect of a compound in an animal model of aparticular disorder such as cancer.

Suitable cell lines for subcutaneous xenograft experiments include C6cells (glioma, ATCC #CCL 107), A375 cells (melanoma, ATCC #CRL 1619),A431 cells (epidermoid carcinoma, ATCC #CRL 1555), Calu 6 cells (lung,ATCC #HTB 56), PC3 cells (prostate, ATCC #CRL 1435), SKOV3TP5 cells andNIH 3T3 fibroblasts genetically engineered to overexpress EGFR, PDGFR,IGF-1R or any other test kinase. The following protocol can be used toperform xenograft experiments:

Female athymic mice (BALB/c, nu/nu) are obtained from SimonsenLaboratories (Gilroy, Calif.). All animals are maintained underclean-room conditions in Micro-isolator cages with Alpha-dri bedding.They receive sterile rodent chow and water. ad libitum.

Cell lines are grown in appropriate medium (for example, MEM, DMEM,Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2 mMglutamine (GLN)). All cell culture media, glutamine, and fetal bovineserum are purchased from Gibco Life Technologies (Grand Island, N.Y.)unless otherwise specified. All cells are grown in a humid atmosphere of90-95% airand 5-10% CO₂ at 37° C. All cell lines are routinelysubcultured twice a week and are negative for mycoplasma as determinedby the Mycotect method (Gibco).

Cells are harvested at or near confluency with 0.05% Trypsin-EDTA andpelleted at 450×g for 10 min. Pellets are resuspended in sterile PBS ormedia (without FBS) to a particular concentration and the cells areimplanted into the hindflank of the mice (8-10 mice per group, 2-10×10⁶cells/animal). Tumor growth is measured over 3 to 6 weeks using veniercalipers. Tumor volumes are calculated as a product oflength×width×height unless otherwise indicated. P values are calculatedusing the Students t-test. Test compounds in 50-100 μL excipient(DMSO,or VPD:D5W) can be delivered by IP injection at different concentrationsgenerally starting at day one after implantation.

Tumor Invasion Model

The following tumor invasion model has been developed and may be usedfor the evaluation of therapeutic value and efficacy of the compoundsidentified to selectively inhibit KDR/FLK-1 receptor.

Procedure

8 week old nude mice (female) (Simonsen Inc.) are used as experimentalanimals. Implantation of tumor cells can be performed in a laminar flowhood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and5 mg/kg Xylazine) are administered intraperitoneally. A midline incisionis done to expose the abdominal cavity (approximately 1.5 cm in length)to inject 10⁷ tumor cells in a volume of 100 μl medium. The cells areinjected either into the duodenal lobe of the pancreas or under theserosa of the colon. The peritoneum and muscles are closed with a 6-0silk continuous suture and the skin is closed by using wound clips.Animals are observed daily.

Analysis

After 2-6 weeks, depending on gross observations of the animals, themice are sacrificed, and the local tumor metastases to various organs(lung, liver, brain, stomach, spleen, heart, muscle) are excised andanalyzed (measurement of tumor size, grade of invasion, immunochemistry,in situ hybridization determination, etc.).

C-Kit Assay

This assay is used to detect the level of c-kit tyrosinephosphorylation.

MO7E (human acute myeloid leukemia) cells were serum starved overnightin 0.1% serum. Cells were pre-treated with the compound (concurrent withserum starvation), prior to ligand stimulation. Cells were stimulatedwith 250 ng/ml rh-SCF for 1.5 minutes. Following stimulation, cells werelysed andimmunoprecipitated with an anti-c-kit antibody. Phosphotyrosineand protein levels were determined by Western blotting.

MTT Proliferation Assay

MO7E cells were serum starved and pre-treated with compound as describedfor the phosphorylation experiments. Cells were plated @ 4×10⁵cells/well in a 96 well dish, in 100 μl RPMI+10% serum. rh-SCF (100ng/mL) was added and the plate was incubated for 48 hours. After 48hours, 10 μl of 5 mg/ml MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added and allowed to incubate for 4 hours. Acidisopropanol (100 μl of 0.04N HCl in isopropanol) was added and theoptical density was measured at a wavelength of 550 nm.

Apoptosis Assay

MO7E cells were incubated ±SCF and ±compound in 10% FBS withrh-GM-CSF(long/mL) and rh-IL-3 (10 ng/mL). Samples were assayed at 24and 48 hours. To measure activated caspase-3, samples were washed withPBS and permeabilized with ice-cold 70% ethanol. The cells were thenstained with PE-conjugated polyclonal rabbit anti-active caspase-3 andanalyzed by FACS. To measure cleaved PARP, samples were lysed andanalyzed by western blotting with an anti-PARP antibody.

Additional Assays

Additional assays which may be used to evaluate the compounds of thisinvention include, without limitation, a bio-flk-1 assay, an EGFreceptor-HER2 chimeric receptor assay in whole cells, a bio-src assay, abio-lck assay and an assay measuring the phosphorylation function ofraf. The protocols for each of these assays may be found in U.S.application Ser. No. 09/099,842, which is incorporated by reference,including any drawings, herein.

Measurement of Cell Toxicity

Therapeutic compounds should be more potent in inhibiting receptortyrosine kinase activity than in exerting a cytotoxic effect. A measureof the effectiveness and cell toxicity of a compound can be obtained bydetermining the therapeutic index, i.e., IC₅₀/LD₅₀. IC₅₀, the doserequired to achieve 50% inhibition, can be measured using standardtechniques such as those described herein. LD₅₀, the dosage whichresults in 50% toxicity, can also be measured by standard techniques aswell (Mossman, 1983, J. Immunol. Methods, 65:55-63), by measuring theamount of LDH released (Korzeniewski and Callewaert, 1983, J. Immunol.Methods, 64:313, Decker and Lohmann-Matthes, 1988, J. Immunol. Methods,115:61), or by measuring the lethal dose in animal models. Compoundswith a large therapeutic index are preferred. The therapeutic indexshould be greater than 2, preferably at least 10, more preferably atleast 50.

B. Example of Cellular Assay Results Using5-(5-Fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide

To confirm the potency of5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) detected in biochemicalassays (vide infra), the ability of said compound to inhibitligand-dependent RTK phosphorylation was evaluated in cell-based assaysusing NIH-3T3 mouse cells engineered to overexpress Flk-1 or humanPDGFRβ.5-(5-Fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) inhibited VEGF-dependentFlk-1 tyrosine phosphorylation with an IC₅₀ value of approximately 0.03μM. This value is similar to the 0.009 μM K_(i) value determined forinhibition of Flk-1 by5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) determined in biochemicalassays. This indicates that5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) readily penetrates intocells. Consistent with the biochemical data (vide infra) indicating that5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) had comparable activityagainst Flk-1 and PDGFRβ, it was also found that it inhibitedPDGF-dependent receptor phosphorylation in cells with an IC₅₀ value ofapproximately 0.03 μM. The ability of5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) to inhibit c-kit, aclosely related RTK that binds stem cell factor (SCF), was determinedusing MO7E cells that express this receptor. In these cells,5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) inhibited SCF-dependentc-kit phosphorylation with an IC₅₀ value of 0.01-0.1 μM. This compoundalso inhibited SCF-stimulated c-kit phosphorylation in acute myeloidleukemia (AML) blasts isolated from the peripheral blood of patients.

In addition to testing the ability of5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) to inhibitligand-dependent receptor phosphorylation in cells, its effect onligand-dependent proliferative response of cells was also examined invitro (see Table 4). In these studies, cells quiesced by overnight serumstarvation were induced to undergo DNA synthesis upon addition of theappropriate mitogenic ligand. As shown in Table 4,5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) inhibited thePDGF-induced proliferation of NIH-3T3 cells overexpressing PDGFRβ orPDGFRα with IC₅₀ values 0.031 and 0.069 μM, respectively, and theSCF-induced proliferation of MO7E cells with an IC₅₀ value of 0.007 μM.TABLE 4 Cellular IC₅₀ Biochemical Receptor Ligand-dependent K_(i) ¹Phosphorylation Proliferation Receptor (μM) (μM) (μM) Flk-1/KDR 0.0090.03² 0.004³ PDGFRα 0.008 0.03⁴ 0.031⁴ PDGFRβ ND ND 0.069⁵ FGFR 0.83 ND0.7³ c-kit ND 0.01-0.1⁶ 0.007⁶ND = Not Determined¹Determined using recombinant enzyme²Determined usinq serum-starved NIH-3T3 cells expressing Flk-1³Determined using serum-starved HUVECs⁴Determined using serum-starved NIH-3T3 cells expressing PDGFR□⁵Determined using serum-starved NIH-3T3 cells expressing PDGFR□⁶Determined using serum-starved MO7E cells

As shown in Table 4, there is a general agreement between thebiochemical and cellular activities of5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) supporting the conclusionthat this compound crosses cellular membranes. Further, it can beconcluded that the cellular responses are a result of the activity ofcompound 80 against the indicated target. In contrast, when tested inthe presence of complete growth medium in vitro, substantially higherconcentrations of5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) (>10 μM) were required toinhibit the growth of a variety of human tumor cells (see Table 5). Thisindicates that the compound did not directly inhibit the growth of thesecells at concentrations required to inhibit ligand-dependent receptorphosphorylation and cell proliferation. TABLE 5 Cell IC₅₀ LD₅₀ LineOrigin (μM) (μM) HT29 Colon carcinoma 10 22 A549 Lung carcinoma 9.5 22NCI-H460 NSC lung carcinoma 8.9 20 SF767T Glioma 7.9 14 A431 Epidermoidcarcinoma 6.0 18

Briefly, the results shown in Table 5 were obtained by incubating cellsfor 48 hr in complete growth medium in the presence of serial dilutions5-(5-Fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide. At the end of the growth period, therelative number of cells was determined. IC₅₀ values were calculated asthe concentration of compound that inhibited the growth of cells by 50%relative to untreated cells. LD₅₀ values were calculated as theconcentration of compound that caused a 50% reduction in the number ofcells relative to those at the start of the experiment.

A more relevant cell-based assay in which to evaluate theanti-angiogenic potential of5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) is the in vitromitogenesis assay using human umbilical vein endothelial cells (HUVECs)as a model system for the endothelial cell proliferation critical to theangiogenic process. In this assay, a mitogenic response, measured as anincrease in DNA synthesis, is induced in serum-starved HUVECs uponaddition of VEGF or FGF. In these cells,5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) inhibited the VEGF- andFGF-induced mitogenic response in a dose-dependent manner with IC₅₀values of 0.004 μM and 0.7 μM, respectively, when compound was presentthroughout the 48-hr assay.

Briefly, the aforementioned results were obtained using Serum-starvedHUVECs that were incubated with mitogenic concentrations of VEGF (100ng/ml) or FGF (30 ng/ml) in the presence of serial dilutions of5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) for 24 hrs. The mitogenicresponse during the following 24 hrs. in the presence of ligand andinhibitor was quantitated by measurement of DNA synthesis based onincorporation of bromodeoxyuridine into cellular DNA.

In separate experiments, compound 80 inhibited the VEGF-dependentphosphorylation of ERK 1/2 (p42/44MAP kinase), an early downstreamtarget of Flk-1/KDR, in a dose-dependent manner. The inhibitory activityof compound 80 was also shown to be long-lasting in this system;inhibiting VEGF-dependent phosphorylation of ERK 1/2 for as long as 48hours after removal of5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) from the medium followinga short (2 hr) exposure to micromolar concentrations of the compound.

VEGF has been recognized to be an important survival factor forendothelial cells. Since5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) inhibits theVEGF-dependent mitogenic response of HUVECs, the effect of the compoundon HUVEC survival was investigated. In these experiments, cleavage ofthe caspase 3 substrate poly-ADP-ribosyl polymerase (PARP) was used as areadout for apoptosis. HUVECs cultured in serum-free conditions for 24hours exhibited substantial levels of PARP cleavage, as detected by theaccumulation of the 23 kDa PARP cleavage fragment. This was largelyprevented by the addition of VEGF to the cell medium, indicating thatVEGF acts as a survival factor in this assay.5-(5-Fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) has been shown to inhibitKDR signaling. Accordingly,5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) inhibited VEGF-mediatedHUVEC survival in a dose-dependent manner. Thus, these data indicatethat compound 80 induced apoptosis in endothelial cells in culture inthe presence of VEGF.

C. In Vivo Efficacy Studies

i. Efficacy Against Established Tumor Xenografts

The in vivo efficacy of5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) was studied insubcutaneous (SC) xenograft models using human tumor cells implantedinto the hindflank region of athymic mice. Following implantation,tumors were allowed to become established to a size of 100-550 mm³ priorto starting oral treatment with the compound.

Daily oral administration of compound 80 caused a dose-dependentinhibition of A431 tumor growth when treatment was initiated aftertumors had grown to a size of 400 mm³. Statistically significant(P<0.05) inhibition of tumor growth was seen at doses of 40 mg/kg/day(74% inhibition) and 80 mg/kg/day (84% inhibition) (see Table 6). Inpreliminary experiments, a higher (160 mg/kg/day) dose of the compoundwas not more efficacious against established A431 tumors than the 80mg/kg/day dose. In addition, mice treated at the 160 mg/kg/day dose ofthe compound lost body weight, indicating that the higher dose was notas well tolerated. Similar results were obtained in an experiment inwhich A431 tumors were only allowed to reach 100 mm³ in size (see Table5). In this second experiment, complete regression of the tumorsoccurred in six of the eight animals treated at the 80 mg/kg/day for 21days. In these six animals, the tumors did not regrow during a 110-dayobservation period following the end of treatment. In the two animals inwhich the tumors regrew to a large size (2000-3000 mm³), the tumorsregressed in response to a second round of treatment with compound 80.Importantly, in all efficacy experiments,5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) at 80 mg/kg/day has beenwell tolerated, even when dosed continuously for more than 100 days.TABLE 6 % Initial Tumor Compound¹ Inhibition Volume (mm³) (mg/kg/day)(day) P-Value 400 80 84 (36) 0.001 40 74 (36) 0.003 20 51 (36) 0.130 10080 93 (40) 0.002 40 75 (40) 0.015 10 61 (40) 0.059¹Compound 80.

Briefly, the results shown in Table 6 were obtained using A431 cells(0.5×106 cells/mouse) which were implanted SC into the hindflank regionof athymic mice. Daily oral administration of5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) in a Cremophore-basedvehicle or vehicle control began when tumors reached the indicatedaverage volume. Tumors were measured using vernier calipers and tumorvolume was calculated as the product of length x width x height.F-values were calculated by comparing the size of the tumors for animalsthat were treated with compound 80 (n=8) to those of animals that weretreated with a vehicle (n=16) on the last day of the experiment, usingthe two-tailed Student's t-test.

The efficacy compound 80 against established human tumors of differentorigins was determined using Colo205 (colon carcinoma), SF763T (glioma),and NCI-H460 (non-small cell lung carcinoma) xenografts (see Table 7).These experiments were conducted using5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) administered orally at 80mg/kg/day; a dose that was effective and well tolerated. TABLE 7 %Initial Tumor Inhibition Tumor Type Volume (mm³) (day) P-Value A431¹Epidermoid 100 93 (40) 0.002 A431¹ Epidermoid 400 84 (36) 0.001 Colo205Colon 370 77 (54) 0.028 NCI-H460 Lung 300 61 (54) 0.003 SF763T Glioma550 53 (30) 0.001¹Data are from experiment reported in Table 5.

In the abovementioned experiments, compound 80 was administered oncedaily at 80 mg/kg in a Cremophor-based vehicle once tumors reached theindicated size. Percent inhibition compared to the vehicle-treatedcontrol group was calculated at termination of the experiments. P-valueswere calculated by comparing tumor sizes of the animals that had beentreated with the compound to tumor sizes of those animals that had beentreated with the vehicle, using the two-tailed Student's t-test.

Although5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide (Compound 80) inhibited the growth ofall the tumor types shown in Table 7, there was a difference in theresponse of the different xenograft models. Specifically, the growth ofNCI-H460 and SF763T tumors was arrested or greatly slowed whereas theColo205 tumors, like A431 tumors, regressed when treated with5-(5-Fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide.

In order to determine the molecular basis for the difference in responsebetween xenograft models, the SF763T tumors were studied. Therefore,SF763T tumors, which were less responsive-to treatment with5-(5-Fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)amide, have been evaluated at the molecularlevel using immunohistological-techniques to determine the effect oftreatment with the compound. These studies were initially conducted inthis tumor type because SF763T tumors are highly vascularized withmicrovessels that strongly express the endothelial cell marker CD31 andare hence well suited for studies of tumor microvessel density (MVD).Immunohistological evaluation of SF763T tumors indicated that tumorsfrom treated animals had reduced MVD relative to vehicle-treatedcontrols, consistent with an anti-angiogenic mechanism of action forcompound 80; MVD was 24.2±4.1 in animals treated with compound 80,compared to 39.3±5.7 for those that were treated with just the vehicle.As anticipated from the associated tumor growth arrest, a pronouncedinhibition of tumor cell proliferation was evident in tumors that weretreated with compound 80. These tumors had half the mitotic index ofthose in vehicle-treated tumors (data not shown). The effect compound 80on MVD and tumor cell proliferation indicates that the compound hasprofound anti-angiogenic and anti-tumor effects, even under conditionsin which tumors do not regress.

The ability of compound 80 to inhibit PDGFR phosphorylation andsubsequent signaling in vivo was also evaluated in the SF763T tumors,which express high levels of PDGFRP. Treatment of the SF763T tumors withcompound 80 strongly inhibited PDGFRP tyrosine phosphorylation inestablished SF763T tumors. Compound 80 also reduced the levels ofphosphorylated (activated) phospholipase C gamma (PLC-γ), an immediatedownstream indicator of PDGFR activation. These data demonstrate thatoral administration of compound 80 causes a direct effect on target(PDGFR) activity in tumors in vivo.

Based on the demonstration that the ability of compound 80 to inhibitVEGF-dependent signaling in HUVECs in vitro was long-lasting (videsupra), the efficacy of the compound was evaluated when the compound wasadministered infrequently in the Colo205 tumor model. As shown in Table8, 80 mg/kg (91% inhibition) and 40 mg/kg (84% inhibition) wereefficacious when administered daily, but not when administered twiceweekly. In contrast, a higher dose of compound 80 (160 mg/kg) didinhibit (52% inhibition) the growth of established Colo205 tumors whenadministered twice weekly, suggesting that this compound can demonstrateefficacy when administered infrequently at a higher dose. It should benoted that dosing regimens may be determined by those with ordinaryskill in the art without undue experimentation. TABLE 8 Dose (mg/kg)Frequency % Inhibition P-Value 160 Twice weekly 52 0.085 Once weekly 17NS 80 Daily 91 0.039 Twice weekly 19 NS Once weekly 0 NS 40 Daily 840.028 Twice weekly 36 NSNS: not significant (P > 0.05)

Briefly, the results shown in Table 8 were obtained using Colo205 cells(0.5×10⁶ cells/mouse) that had been implanted SC into the hindflankregion of athymic mice. Oral administration of compound 80 according tothe indicated schedule began when tumors reached 400 mm³. Tumors were,measured using vernier calipers and tumor volume was calculated as theproduct of length×width×height. P-values were calculated by comparingthe size of the tumors for animals that were treated with compound 80 tothose of animals that were treated with a vehicle on the last day of theexperiment, using the two-tailed Student's t-test.

ii. Efficacy of Compound 80 in a Model of Disseiminated Disease

In addition to supporting the sustained growth of solid primary tumors,angiogenesis is also an essential component supporting the developmentof disseminated disease due to metastasis from the primary tumor. Theeffect of compound 80 on the development of disseminated disease wasexamined in the B16-F1 mouse melanoma lung colonization model. In thismodel, B16-F1 cells inoculated intravenously via the tail vein ofathymic mice colonize the lungs and form tumors. As shown in Table 8,oral administration of compound 80 at 80 mg/kg/day effectively reducedthe burden of B16-F1 cells in the lung as evaluated by measurements oftotal lung weight. These data suggest that compound 80 can inhibitdisseminated disease in vivo. TABLE 9 Lung Weight (g) % InhibitionP-Value Vehicle 0.83 ± 0.07 — — Compound¹ 0.41 ± 0.04 50 <0.001¹Compound 80

Briefly, the results shown in Table 9 were obtained using athymic micethat had been inoculated with B16-F1 tumor cells (5×10⁵ cells/mouse) viathe tail vein. Mice were treated daily with orally administered compound80 at 80 mg/kg/day (n=10) or vehicle (n=18) for 24 days after tumor cellinoculation. At the end of the treatment period, the mice weresacrificed and their lungs removed and weighed. Percent inhibition wascalculated by comparing the lung weight of those animals that had beentreated with compound 80, with the lung weight of the animals that hadonly been treated with vehicle. P-values were determined using thetwo-tailed Student's t-test.

D. Examples of Biological Activity

Examples of the in vitro potency of compounds of this invention areshown in Table 2.

CONCLUSION

In studies to investigate the pharmacokinetic characteristics of thecompounds of the preferred embodiments of the present invention it hasbeen demonstrated that oral administration of a single dose of saidcompounds resulted in high oral bioavailability in mice. The good oralbioavailability and linear pharmacokinetics indicate that the compoundsof the preferred embodiments of the present have favorablepharmacokinetic characteristics.

In addition, the compounds of the preferred embodiments of the presentinvention are potent inhibitor of the tyrosine kinase activity of thesplit-kinase domain RTKs Flk-1/KDR and PDGFR, which are involved inangiogenesis, and the RTK c-kit, a receptor for stem cell factor (SCF),that is involved in certain hematologic cancers. At higherconcentrations, the compounds of the preferred embodiments of thepresent invention also inhibit the tyrosine kinase activity of FGFR-1, athird RTK involved in angiogenesis. Consistent with their biochemicalactivity, the compounds of the preferred embodiments of the presentinvention inhibit the ligand-dependent tyrosine phosphorylation oftarget RTKs and the in vitro mitogenic response of human umbilical veinendothelial cells (HUVECs) stimulated with VEGF or FGF, ofPDGFR-expressing NIH-3T3 cells stimulated with PDGF, and of MO7E acutemyeloid leukemia cells stimulated with SCF. In contrast, the compoundsof the preferred embodiments of the present invention do not directlyinhibit the proliferation of tumor cells in complete growth mediumexcept at concentrations 2 to 3 orders of magnitude higher than thoserequired to inhibit the ligand-dependent mitogenic responses. In mousexenograft studies, the compounds of the prefered embodiments of thepresent invention inhibited the growth of established human tumors ofvarious origins in a dose-dependent manner and at concentrations thatwere well tolerated even upon extended (>100 days) dosing. At 80mg/kg/day, the compounds of the preferred embodiments of the presentinvention induced regression of large established A431 and Colo205tumors, and caused substantial growth inhibition or stasis of SF763T andNCI-H460 tumors. In mice bearing SF763T tumors, the compounds of thepreferred embodiments of the present invention caused reductions inmicrovessel density, phosphorylation of PDGFR in the tumors, and mitoticindex in the tumor cells. At this dose, the compounds of the preferredembodiments of the present invention also inhibited lung colonization byB16-F1 tumor cells in a model of tumor metastasis. Regimen studiesdemonstrated that the compounds of the preferred embodiments of thepresent invention are most efficacious when administered daily. Directevidence of the anti-angiogenic activity of the compounds of thepreferred embodiments of the present invention was detected in SF763Ttumors in which microvessel density was reduced. Direct evidence thatthe compounds of the preferred embodiments of the present inventioninhibit PDGFR phosphorylation and signaling in vivo was also obtained inSF763T tumors.

Taken together, these data support the notion that orally administeredcompounds of the preferred embodiments of the present invention areanti-angiogenic agents for the treatment of cancers, including solidtumors and hematological malignancies in which angiogenesis and/orsignaling through c-kit are important in the disease pathology.

It will be appreciated that the compounds, methods and pharmaceuticalcompositions of the present invention are effective in modulating PKactivity and therefore are expected to be effective as therapeuticagents against RTK, CTK-, and STK-related disorders.

One skilled in the art would also readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent herein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. For example, if X isdescribed as selected from the group consisting of bromine, chlorine,and iodine, claims for X being bromine and claims for X being bromineand chlorine are fully described.

Other embodiments are within the following claims.

1-60. (canceled)
 61. A compound of Formula (I);

wherein: R¹ is selected from the group consisting of hydrogen, halo,alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,—(CO)R¹⁵, —NR¹³R¹⁴, —(CH₂)_(r)R¹⁶ and —C(O)NR⁸R⁹; R² is selected fromthe group consisting of hydrogen, halo, alkyl, trihalomethyl, hydroxyalkoxy, —NR¹³R¹⁴, —NR¹³C(O)R¹⁴, —C(O)R¹⁵, aryl, heteroaryl, and—S(O)₂NR¹³R¹⁴; R³ is selected from the group consisting of hydrogen,halogen, alkyl, trihalomethyl, hydroxy, alkoxy, —C(O)R¹⁵, —NR¹³R¹⁴,aryl, heteroaryl, —NR¹³S(O)₂R¹⁴, —S(O)₂NR¹³R¹⁴, —NR¹³C(O)R¹⁴, and—NR¹³C(O)OR¹⁴; R⁴ is selected from the group consisting of hydrogen,halogen, alkyl, hydroxy, alkoxy and —NR¹³R¹⁴; R⁵ is selected from thegroup consisting of hydrogen and alkyl; R⁶ is —C(O)R¹⁰; R⁷ is selectedfrom the group consisting of hydrogen, alkyl, aryl and heteroaryl; R⁸and R⁹ are independently selected from the group consisting of hydrogen,alkyl and aryl; R¹⁰ is —N(R¹¹)(CH₂)_(n)R²; R¹¹ is selected from thegroup consisting of hydrogen and alkyl; R¹² is selected from the groupconsisting of —NR¹³R¹⁴, hydroxy, —C(O)R¹⁵, aryl, and heteroaryl; R¹³ andR¹⁴ are independently selected from the group consisting of hydrogen,alkyl, cycloalkyl, aryl and heteroaryl; or R¹³ and R¹⁴ may combine toform a group selected from the group consisting of —(CH₂)₄—, —(CH₂)₅—,—(CH₂)₂O(CH₂)₂—, and —(CH₂)₂N(CH₃)(CH₂)₂; R¹⁵ is selected from the groupconsisting of hydrogen, hydroxy, alkoxy and aryloxy; R¹⁶ is selectedfrom the group consisting of hydroxy, —C(O)R¹⁵, —NR¹³R¹⁴ and—C(O)NR¹³R¹⁴; R¹⁷ is selected from the group consisting of alkyl,cycloalkyl, aryl and heteroaryl; and n and r are independently 1, 2, 3,or 4; or a pharmaceutically acceptable salt thereof
 62. The compound ofclaim 61, wherein R¹ is selected from the group consisting of hydrogen,lower alkyl, —(CH₂)_(r)R¹⁶ and —C(O)NR⁸R⁹; R² is selected from the groupconsisting of hydrogen, halogen, aryl and —S(O)₂NR¹³R¹⁴; R³ is selectedfrom the group consisting of hydrogen, lower alkyl, lower alkoxy, aryl,heteroaryl, and —C(O)R¹⁵; R⁴ is hydrogen; R⁵ is selected from the groupconsisting of hydrogen and lower alkyl; R⁷ is selected from the groupconsisting of hydrogen, lower alkyl, and aryl; R¹⁶ is selected from thegroup consisting of hydroxy and C(O)R¹⁵; and r is 2 or
 3. 63. Thecompound of claim 62, wherein n is 1, 2, or 3; R¹¹ is hydrogen; and R¹²is selected from the group consisting of hydroxy, lower alkoxy, C(O)R¹⁵,heteroaryl and —NR¹³R¹⁴.
 64. The compound of claim 63, wherein R¹³ andR¹⁴ are independently selected from the group consisting of hydrogen,lower alkyl, heteroaryl and, combined, —(CH)₄—, —(CH₂)₅—,—CH₂)₂—O—(CH₂)₂— and —(CH₂)₂N(CH₃)(CH₂)₂—.
 65. A compound of formula

or a pharmaceutically acceptable salt thereof.
 66. A compound that isthe L-malate salt of5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylaminoethyl)amide.
 67. A compound of formula

or a pharmaceutically acceptable salt thereof.
 68. A pharmaceuticalcomposition comprising a compound of claim 61 and a pharmaceuticallyacceptable carrier or excipient.
 69. A pharmaceutical compositioncomprising the compound of claim 65 and a pharmaceutically acceptablecarrier or excipient.
 70. A pharmaceutical composition comprising thecompound of claim 66 and a pharmaceutically acceptable carrier orexcipient.
 71. A pharmaceutical composition comprising the compound ofclaim 67 and a pharmaceutically acceptable carder or excipient.